The Study of Microbial Structure:

Microscopy and Specimen Preparation

• Microscope: Microbiology deals with almost

all those organism which cannot be seen with
naked eye.
• Microscope is crucial.
• Components of the microscope are also
important to understand

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Lenses
 A lens is an optical device which transmits
and refracts light, converging or diverging the
beam.
 Refraction
 When a ray of light passes from one medium
to another, it bends, this is called refraction.
 Refractive index
 a measure of how greatly a substance slows the
velocity of light
 direction and magnitude of bending is
determined by the refractive indices of the
two media forming the interface
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• Focal Point
• Focus light rays at a specific place called the
focal point
• Focal length
• Distance between center of lens and focal
point is the focal length
• strength of lens related to focal length
– short focal length more magnification

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 Types of Microscopes
Light Microscope
bright-field microscope
dark-field microscope
phase-contrast microscope
fluorescence microscope

Electron Microscope
Transmission Electron Microscope
Scanning Electron Microscope

Newer Techniques in Microscopy

Confocal scanning laser (CLSM) microscopy
Scanning probe microscope

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 The Bright-Field Microscope(BFM)
• produces a dark image against a brighter
background
• has several objective lenses
– BFM microscopes remain in focus when
objectives are changed
• total magnification
– product of the magnifications of the ocular
lenses and the objective lenses

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Microscope Resolution
• ability of a lens to separate or distinguish
small objects that are close together
• wavelength of light used is major factor in
resolution
It can also be explained by an equation called
Abbe equation

shorter wavelength  greater resolution

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Working distance
— distance between the front surface of lens and surface of cover glass or specimen
when it is in sharp focus

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 The Dark-Field Microscope
• Image is formed by light reflected or refracted by
specimen
• produces a bright image of the object against a
dark background
• used to observe living, unstained preparations
– For eukaryotes has been used to observe internal
structures
– For prokaryotes has been used to identify bacteria
such as Treponema pallidum, the causative agent of
syphilis

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 The Phase-Contrast Microscope
• enhances the contrast between
intracellular structures having slight
differences in refractive index
• excellent way to observe living cells
– Especially useful for detecting bacterial
components such as endospores and inclusion
bodies that have refractive indices different
from that of water

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 The Differential Interference Contrast
Microscope (DIC)
• creates image by detecting differences in
refractive indices and thickness of different
parts of specimen
• excellent way to observe living cells
– Live, unstained cells appear brightly colored and
three-dimensional

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 The Fluorescence Microscope
• exposes specimen to ultraviolet, violet, or blue
light
• specimens usually stained with fluorochromes
(A fluorescent chemical)
• Shows a bright image of the object resulting
from the fluorescent light emitted by the
specimen
• Has applications in medical microbiology and
microbial ecology studies

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 Preparation and Staining of Specimens
• increases visibility of specimen
• highlight specific morphological features
• preserves specimens
A: Fixations
 preserves internal and external structures and
fixes them in position
 organisms usually killed and firmly attached to
microscope slide
 heat fixation – routine use with prokaryotes
 preserves overall morphology but not internal structures
 chemical fixation – used with larger, more delicate
organism, protects fine cellular substructure and morphology
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 B: Dyes and Simple Staining
• Dyes
– make internal and external structures of cell more
visible by increasing contrast with background
– have two common features
• chromophore groups
– chemical groups with conjugated double bonds
– give dye its color
• ability to bind cells
– Ionizable dyes have charged groups
• basic dyes have positive charges
• acid dyes have negative charges
• Simple stains
– a single stain is used
– use can determine size, shape and arrangement of 18
bacteria
 Differential Staining
• divides microorganisms into groups based
on their staining properties
 e.g., Gram stain
 e.g., acid-fast stain
Gram staining
most widely used differential staining procedure
divides bacteria into two groups based on differences in cell wall structure

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 Acid-fast staining
• particularly useful for staining members of
the genus Mycobacterium
e.g., Mycobacterium tuberculosis – causes tuberculosis
e.g., Mycobacterium leprae – causes leprosy
– high lipid content in cell walls is responsible for
their staining characteristics
Staining Specific Structures
 negative staining
 e.g., capsule stain used to visualize capsules
surrounding bacteria
 capsules are colorless against a stained
background
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• endospore staining
– double staining technique
– bacterial endospore is one color and vegetative
cell is a different color
• flagella staining
– mordant applied to increase thickness of flagella

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 Electron Microscopy
• beams of electrons are
used to produce images
• wavelength of electron
beam is much shorter
than light, resulting in
much higher resolution

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 The Transmission Electron
Microscope (TEM)
• electrons scatter when they pass through
thin sections of a specimen
• transmitted electrons (those that do not
scatter) are used to produce image
• denser regions in specimen, scatter more
electrons and appear darker

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 Specimen Preparation
• Similar to procedures used for light
microscopy
• for transmission electron microscopy,
specimens must be cut very thin
• specimens are chemically fixed and stained
with electron dense material
• shadowing
– coating specimen with a thin film of a heavy
metal
• freeze-etching
– freeze specimen then fracture along lines of
greatest weakness (e.g., membranes) 29
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 The Scanning Electron Microscope
• uses electrons reflected from the surface of
a specimen to create image
• produces a 3-dimensional image of
specimen’s surface features

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 Newer Techniques in Microscopy
• confocal scanning
laser (CLSM)
microscopy and
scanning probe
microscopy
• have extremely high
resolution

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 Confocal Microscopy
• confocal scanning laser microscope
• laser beam used to illuminate a variety of
planes in the specimen
• computer compiles images created from
each point to generate a 3-dimensional
image
• used extensively to observe biofilms

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 Scanning Probe Microscopy
• A: scanning tunneling microscope
– steady current (tunneling current) maintained
between microscope probe and specimen
– up and down movement of probe as it maintains
current is detected and used to create image of
surface of specimen

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• Atomic force microscope
– sharp probe moves over surface of specimen at
constant distance
– up and down movement of probe as it
maintains constant distance is detected and
used to create image

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CHEERS