ERT107
MICROBIOLOGY
FOR BIOPROCESS
ENGINEERING
Pn Syazni Zainul Kamal
PPK Bioprocess
Chapter 2: Microscopy
Techniques
CO2 :
Ability to demonstrate practices in microscopy,
staining, sterilization, isolation and identification
of bacteria and fungi
Why need to study microscope?
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Microbiology concerned with organisms so
small they cannot be seen distinctly with the
unaided eye
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Thus, it is important to understand how the
microscope works and the way in which
specimens are prepared for examination
Unit of measurement
Microscopy
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1)
2)
3)
4)
Refer to the use of light or electrons to
magnify objects
The same general principles guide both light
and electron microscopy :
Wavelength of Radiation
Magnification of an image
Resolving power of the instrument
Contrast in the specimen
Wavelength of Radiation
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Various forms of radiation differ in wavelength,
which is the distance between two corresponding
parts of a wave.
The human eye distinguishes different wavelengths
of light as different colors. (wvlength of 400nm as
violet)
In addition to light, moving electrons act as waves
with wavelengths dependent upon the voltage of an
electron beam.
Electron wavelengths are much smaller than those of
visible light, and thus their use results in enhanced
microscopy.
Magnification
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Magnification is the apparent increase in size of an
object and is indicated by a number followed by an
“X” which is read “times.”
eg. Cells are magnified 16,000 x or 16,000 times
Magnification results when a beam of radiation
refracts (bends) as it passes through a lens
Light beam slows as it enters glass, and the beam
bends
Light also bends as it leave the glass and reenter the air
Light beam spread apart as they travel past the focal
point and produce enlarged image
Resolution
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Resolution (also called resolving power) is the ability
to distinguish between small objects that are close
together.
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The better the resolution, the better the ability to
distinguish two objects that are close to one another.
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Modern microscopes can distinguish between objects
as close as 0.2 μm.
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A principle of microscopy is that resolution
distance is dependent on
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the wavelength of the electromagnetic radiation
the numerical aperture of the lens, which is its
ability to gather light.
Immersion oil is used to fill the space between
the specimen and a lens to reduce light
refraction and thus increase the numerical
aperture and resolution.
Contrast
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Contrast refers to differences in intensity
between two objects, or between an object and
its background.
Since most microorganisms are colorless, they
are stained to increase contrast.
Polarized light may also be used to enhance
contrast.
Good contrast
Poor contrast
Types of microscope
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Light microscope (use to look at intact cells)
Electron microscope (use to look at internal
structure or details of cell surfaces)
Light microscope
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Types of light microscope :
1) bright-field
2) phase contrast
3) dark-field
4) fluorescence
5) confocal
Bright-Field Microscope
bright-field microscope
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The most common microscope is bright-field
microscope. It forms a dark image against a brighter
background.
There are two basic types of bright-field microscope:
1) Simple microscope (Leeuwenhoek microscope)
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contain a single magnifying lens and are similar to a magnifying
glass.
Capable of approx 300X magnification
2) Compound microscope
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use a series of lenses for magnification.
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Compound microscopes
 use a series of lenses for magnification
 view both stained & unstained sample
 has 4 objective lenses :
- Scanning objective lens (4X),
- low power objective lens (10X),
- high power lens or high dry objective lens
(40X),
- oil immersion objective lens (100X)
 parfocal microscopes remain in focus when
objectives are changed
Light from illuminated specimen focused by
objective lens (enlarged image)
 Ocular lens further magnify the image,
typically another 10 times (10X)
 Total magnification = multiplying the
objective lens & ocular lens magnifications
together
 Eg. Total magnification using 10X ocular lens
and a 10X low power objective lens = 100X
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Microscope resolution
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ability of a lens to separate or distinguish small
objects that are close together
wavelength of light used is major factor in
resolution
shorter wavelength  greater resolution
Immersion oil is used to fill the space between the specimen
and a lens to reduce light refraction and thus increase the
numerical aperture and resolution.
•working distance
— distance between the front surface of lens and surface of cover glass or
specimen when it is in sharp focus
Dark-Field Microscope
Dark-field microscope
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utilize a dark-field stop in the condenser that
prevents light from directly entering the
objective lens.
Only light rays scattered by the specimen enter
the objective lens and are seen
the specimen appears light against a dark
background
used to observe living, unstained preparations
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has been used to observe internal structures in
eukaryotic microorganisms
has been used to identify bacteria such as
Treponema pallidum, the causative agent of
syphilis
ath
Dark-field stop
underneath condenser
Condenser produces a
hollow cone of light so
that the only light
entering the objective
come from specimen
Phase-Contrast Microscope
Phase-Contrast Microscope
 Increase contrast between cell & surrounding
medium
 Cell differ in refractive index from their
surrounding, hence it bend some light ray that
pass through them
 Condenser with anular stop (opaque disk with
thin transparent ring)
 Light passing through unstained cell are retarded
 This effect is amplified by phase ring, leading to
the formation of dark image on light background
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excellent way to observe living cells
 studying microbial motility
 detecting bacterial structures such as
endospores and inclusion bodies that have
refractive indices different from that of
water
The Differential Interference
Contrast Microscope (DIC)
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creates image by detecting differences in
refractive indices and thickness of different
parts of specimen
excellent way to observe living cells
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live, unstained cells appear brightly colored and
three-dimensional
cell walls, endospores, granules, vacuoles, and
nuclei are clearly visible
Fluorescence Microscope
Fluorescence microscope
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developed by O. Shimomuram, M. Chalfie, and R.
Tsien
Since UV light has a shorter wavelength than visible
light, resolution is increased.
Contrast is improved because fluorescing structures
are visible against a black background
Cells stained with fluorescent dyes (fluorochromes)
and bombarded with UV light, they emit visible light
and show up as bright orange, green, yellow or other
colour against black background
has applications in medical microbiology and
microbial ecology studies
Confocal Microscopy
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confocal scanning laser microscopy (CLSM)
creates sharp, composite 3-D image of
specimens by using laser beam, aperture to
eliminate stray light, and computer interface
numerous applications including study of
biofilms