Basic technique in Microbiology

( 102)

MITU AFRIN

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 What is Technique in Microbiology??
• Microbiology Techniques
Microbiology techniques are methods used by microbiologists to study microorganisms, including bacteria,
viruses, fungi, and parasites. These techniques are used to identify, cultivate, and analyze microorganisms
in order to understand their biology, ecology, and potential impact on human health and the environment.
Here are some examples of microbiology techniques:
 Microscopy: Microscopy involves the use of a microscope to view microorganisms. Different types of
microscopes, including compound microscopes, electron microscopes, and fluorescence microscopes, can
be used to study microorganisms at different scales and with different levels of detail.
 Culture techniques: Culture techniques involve the cultivation of microorganisms in the laboratory. This
can be done using various media, including solid and liquid media, and can be used to study the growth,
metabolism, and genetics of microorganisms.
 Molecular techniques: Molecular techniques involve the analysis of the genetic material of
microorganisms, including DNA and RNA. These techniques, such as DNA sequencing and PCR (polymerase
chain reaction), can be used to identify and classify microorganisms, and to study their genetic makeup
and evolution.
 Biochemical techniques: Biochemical techniques involve the analysis of the metabolic products and
enzymes of microorganisms. These techniques, such as enzyme-linked immunosorbent assays (ELISAs) and
biochemical tests, can be used to identify and classify microorganisms and to study their metabolism and
physiology.
Overall, microbiology techniques are essential for understanding the biology of microorganisms and their roles
in various environments and in human health.
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1.(a) What is meant by microscopy? Mention the types
of microscopy.

• Microscopy is the science of investigating small objects and

structures using a microscope.
• Microscopy is the technical field of using microscopes to view
samples & objects that cannot be seen with the unaided
eye (objects that are not within the resolution range of the
normal eye).
• It refers to an optical instrument that uses a lens or an
arrangement of lenses to magnify an object. Also, they help
to view different organisms.

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 Mention the types of microscopy.
• What Are the Different Types of Microscopes?
• There are different types of microscopes and each of these has different
purposes of use. Some are suitable for biological applications, while others
are used in educational institutions. There are also microscope types that
find application in metallurgy and studying three-dimensional samples.
• In this article, there are 5 such microscope types of microscoies . These
five types of microscopes are:
• Light microscope
• Compound microscope
• Electron microscope
• Stereomicroscope
• Scanning probe microscope
• Fluorescent microscope
• X-ray microscope

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 1.(b) Write down the principles of light and electron
microscopy.

• What is a light microscope?

• light microscope is a biology laboratory
instrument or tool, that uses visible light to
detect and magnify very small objects and
enlarge them.
• They use lenses to focus light on the
specimen, magnifying it thus producing an
image. The specimen is normally placed close
to the microscopic lens.

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 Principle of a light microscope (optical
microscope)
• As mentioned earlier, light microscopes visualize an
image by using a glass lens, and magnification is
determined by, the lens’s ability to bend light and focus
it on the specimen, which forms an image.
• When a ray of light passes through one medium into
another, the ray bends at the interface
causing refraction. The bending of light is determined
by the refractive index, which is a measure of how
great a substance slows the speed of light. The
direction and magnitude of the bending of the light are
determined by the refractive indexes of the two
mediums that form the interface.

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 Contd…..
• A medium with a lower refractive index such as glass to air normally
speeds up the light penetration and makes light bend away from
the normal and when light is passed through a medium with a
greater refractive index such as air to glass, it normally slows down
and bends towards the normal, perpendicularly to the surface.
• If an object is put between these two mediums i.e between water
and air, in this case, a prism, the prism will bend the light at an
angle. This is how the microscopic lenses work, they bend the light
at an angle. The lens (convex) on receiving the light rays, focuses
the rays at a specific point known as the focal point (F-point). The
measure of distance from the center of the lens and the focal point
is known as the focal length.
• A microscope uses lenses whose strength is predetermined, in that,
the strength of a lens is directly related to the focal length i.e short
focal length magnifies objects more than lenses with a long focal
length.

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 principles of electron microscopy.
• An electron microscope is a microscope that uses a beam of accelerated
electrons as a source of illumination. It is a special type of microscope
having a high resolution of images, able to magnify objects in nanometres,
which are formed by controlled use of electrons in a vacuum captured on
a phosphorescent screen. Ernst Ruska (1906-1988), a German engineer
and academic professor, built the first Electron Microscope in 1931, and
the same principles behind his prototype still govern modern EMs.

• Principle of electron microscopy

• Electrons are such small particles that, like photons in light, they act as
waves. A beam of electrons passes through the specimen, then through a
series of lenses that magnify the image. The image results from a
scattering of electrons by atoms in the specimen.

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 Working Principle of Electron
microscope
• Electron microscopes use signals arising from the interaction of an electron beam with the sample
to obtain information about structure, morphology, and composition.
• The electron gun generates electrons.
• Two sets of condenser lenses focus the electron beam on the specimen and then into a thin tight
beam.
• To move electrons down the column, an accelerating voltage (mostly between 100 kV-1000 kV) is
applied between the tungsten filament and anode.
• The specimen to be examined is made extremely thin, at least 200 times thinner than those used in
the optical microscope. Ultra-thin sections of 20-100 nm are cut which is already placed on the
specimen holder.
• The electronic beam passes through the specimen and electrons are scattered depending upon the
thickness or refractive index of different parts of the specimen.
• The denser regions in the specimen scatter more electrons and therefore appear darker in the
image since fewer electrons strike that area of the screen. In contrast, transparent regions are
brighter.
• The electron beam coming out of the specimen passes to the objective lens, which has high power
and forms the intermediate magnified image.
• The ocular lenses then produce the final further magnified image.

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• Today there are two major types of electron microscopes
used in clinical and biomedical research settings: the
transmission electron microscope (TEM) and the scanning
electron microscope (SEM); sometimes the TEM and SEM
are combined in one instrument, the scanning transmission
electron microscope (STEM):
• TEM: magnifies 50 to ~50 million times; the specimen
appears flat
• SEM: magnifies 5 to ~ 500,000 times; sharp images of
surface features
• STEM: magnifies 5 to ~50 million times; the specimen
appears flat

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 1(c) Describe the types and applications of light and
electron microscopy.
• Types of light microscopes
1. Bright-field Light Microscope
2. Phase Contrast Light Microscope
3. Dark Field Light Microscope
4. Fluorescent Light Microscope

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 1. Bright-field Light Microscope

• Also acknowledged as a compound light microscope, it is

one of the simplest optical microscopy techniques. The
standard microscopy is used for daily research of simple
diagnostics and can accurately create a dark image against
a white background. It is used to perceive living cells or
fixed specimens. The bright-field light microscope is made
of two lenses- an eyepiece and an objective lens (made
with six or more glasses). With the correct usage, a high-
resolution image is produced.
• The application of a bright-field light microscope is widely
used in microbiology. It can be utilised to view live and
fixed specimens which have been stained. Basic bacteria
cells can be identified easily.

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 2. Phase Contrast Light Microscope

• Phase-contrast light microscopy is a technique used to

make unstained living cells visible. These biological
specimens practically absorb no light, due to which they
may appear transparent with a brightfield microscope.
These cells will have a rich view with phase-contrast by
appearing illuminated. The principle of this type of light
microscopy works on transforming a specimen into an
amplitude image. Once that’s done, it can be clearly
viewed.
• Application of a phase-contrast light microscope is used to
identify animal and plant cells. Microbial elements such as
bacteria endospores can also be detected. The technique is
also widely used to discern the living cells in tissue culture
and keep a check on the growth.

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 3. Dark Field Light Microscope
• The dark field light microscope is used to illuminate living
unstained cells. It is similar to a phase-contrast microscope
which makes the sample brightly lit against a dark
background, and that’s how the name dark field is derived.
Most of the light is reflected away from the camera. This
type of light microscopy places a condenser lens and a
darkfield stop to scatter the beam of light entering the
specimen’s objective. To do so, an opaque disc is set down
under the condenser lens.
• Application of a dark field light microscope is used to view
the internal organs of large cells and identify distinctive
shapes. The technique helps study marine organisms such
as insects, algae and yeast.

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 4. Fluorescent Light Microscope

• A fluorescent light microscope is thoroughly different from those

mentioned earlier. The aforestated types of light microscopy create
an image once the light is transmitted through the cells. The
fluorescent light microscope sheds light by adding a dye molecule
to the specimen. It uses a high-intensity light source and stimulates
the dye molecule when absorbs light energy. The trapped energy is
then released as light. The liberated illuminated energy has a long
wavelength and thereby produces a fluorochrome labelled image.
The microscope uncovers the cells to the light to form an image.
• Application of a fluorescent light microscope is used to view
bacteria cells and identify particular antibodies against bacterial
pathogens. The fluorescent microscope also recognises and
observes microorganisms and identifies the difference between
living and dead bacteria.

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 Types of electron microscopes

• There are two types of electron microscopes,

with different operating styles:

1. Transmission Electron Microscope (TEM)

2. Scanning Electron Microscope (SEM)
3. Environmental Scanning Electron Microscopy (ESEM)
4. Scanning Transmission Electron Microscopy (STEM)
5. Cryo-Electron Microscopy (Cryo-EM)

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 Transmission Electron Microscope
(TEM)
• A TEM generates an electron beam that is focused onto
a narrow sample, often one that is less than 100
nanometers thick. The sample’s atoms are affected by
the electrons, and a detector on the sample’s other
side collects the transmitted electrons.
• The sample is then imaged using the transmitted
electrons, giving high-resolution details on its interior
structure. The crystal structure of materials, the
configuration of atoms and molecules, and even the
positions of individual atoms, may all be seen in TEM
images.

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 Detail on Scanning Electron
Microscopy (SEM)
• A SEM generates an electron beam that is directed onto a
sample’s surface, which is often coated with a thin coating
of metal to improve conductivity. Secondary electrons are
released from the sample’s surface as a result of the
electrons’ interactions with the sample’s atoms. A detector
then picks up these secondary electrons and creates a
picture of the sample.
• SEM images have the potential to be quite detailed,
displaying the sample’s topography down to the nanoscale
level. Due to the fact that various components might result
in a variety of signals in the detector, images can also
provide information regarding the sample’s chemical
makeup.

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 Environmental Scanning Electron
Microscopy (ESEM)
• Environmental scanning electron microscopy
(ESEM) expands the boundaries of traditional
SEM to deliver deeper insights into all types of
samples. ESEM allows for imaging of samples
with minimal preparation and adds variables such
as hydration, thermal cycling, and the
introduction of gas to characterize in situ dynamic
changes. Using water vapor and a temperature
control stage, some of the “impossible to image
samples” such as dirty (highly outgassing) and
naturally hydrated samples (whose properties will
change with drying) are now easily characterized.
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 Scanning Transmission Electron
Microscopy (STEM)
• Scanning transmission electron microscopy
(STEM) is a hybrid electron microscopy
technique used for imaging and morphological
characterization with atomic-scale resolution.
STEM is available on both Covalent's FIB-SEM
instruments, as well as our TEM. Focused-ion-
beam scanning electron microscopes (FIB-
SEMs)

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 Cryo-Electron Microscopy (Cryo-EM)
• Cryo-EM uses frozen samples, gentler electron beams
and sophisticated image processing to overcome these
problems.
• X-ray diffraction can give very high resolution
structures of biomolecules, and several Nobel
prizes have been awarded for just that. But to get an x-
ray structure, you need to be able to crystallise the
molecule. Many proteins won’t crystallise at all. And in
some cases, the process of crystallisation alters the
structure, so it’s not representative of what the
molecule looks like in real life.

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 Cryo-Electron Microscopy (Cryo-EM)
• Cryo-EM doesn’t require crystals, and it also
enables scientists to see how biomolecules
move and interact as they perform their
functions, which is much more difficult using
crystallography.

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 Application of Light Microscopy in
Biology
• Light microscopes play a mammoth part in today’s biology. Here’s how the
application of it is obliging.
• Biologists can detect details at a cellular level and also comprehend about building
blocks of organisms.
• With a light microscope, real-time movements of organisms can be noticed.
• Biologists can also study life-threatening diseases like AIDS and cancer to find a
cure.
• In student life, one can understand the real cells and the supporting structure.
• Mingeraoligsts also make use of light microscopy for sample preparations.
• The technique is easy to learn but hard to master in the medical field.
• Light microscopy is used in pathology labs to identify any disease.
• It can inspect the space and time related to the dynamics of molecules.
• Small elements like nanostructures can be identified easily.
• The application of light microscopy is also useful in metallurgy, chemistry and
gemmology.
• It is widely used in tissue culture research for cell testing.

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 Applications of Electron microscope

• Electron microscopes are used to investigate the ultrastructure of a

wide range of biological and inorganic specimens including
microorganisms, cells, large molecules, biopsy samples, metals, and
crystals.
• Industrially, electron microscopes are often used for quality control
and failure analysis.
• Modern electron microscopes produce electron micrographs using
specialized digital cameras and frame grabbers to capture the
images.
• The science of microbiology owes its development to the electron
microscope. The study of microorganisms like bacteria, virus, and
other pathogens have made the treatment of diseases very
effective.
Thanks

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