The Study of Microbial Structure:
Microscopy and Specimen
Preparation
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Scale
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Discovery of Microorganisms
• Antony van
Leeuwenhoek
(1632-1723)
– first person to
observe and
describe
micro-
organisms Figure 1.1b
accurately
3
Lenses and the Bending of Light
• light is refracted (bent) when passing from
one medium to another
• refractive index
– a measure of how greatly a substance slows
the velocity of light
• direction and magnitude of bending is
determined by the refractive indexes of the
two media forming the interface
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Lenses
• focus light rays at a specific place called the
focal point
• 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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Figure 2.2
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The Light Microscope
• many types
– bright-field microscope
– dark-field microscope
– phase-contrast microscope
– fluorescence microscopes
• are compound microscopes
– image formed by action of 2 lenses
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The Bright-Field Microscope
• produces a dark image against a brighter
background
• has several objective lenses
– parfocal microscopes remain in focus when
objectives are changed
• total magnification
– product of the magnifications of the ocular lens
and the objective lens
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Figure 2.3
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Figure 2.4
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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
shorter wavelength greater resolution
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• working distance
— distance between the front surface of lens and surface of
cover glass or specimen
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Figure 2.5
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Figure 2.6
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The Dark-Field Microscope
• produces a bright image of the object against
a dark background
• used to observe living, unstained preparations
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Figure 2.7b
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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
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Figure 2.9
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Figure 2.10
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The Differential Interference Contrast
Microscope
• 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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The Fluorescence Microscope
• exposes specimen to ultraviolet, violet, or blue
light
• specimens usually stained with fluorochromes
• shows a bright image of the object resulting
from the fluorescent light emitted by the
specimen
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Figure 2.12
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Figure 2.13c and d
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Preparation and Staining of Specimens
• increases visibility of specimen
• accentuates specific morphological features
• preserves specimens
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Fixation
• process by which internal and external
structures are preserved and fixed in
position
• process by which organism is killed and
firmly attached to microscope slide
– heat fixing
• preserves overall morphology but not internal
structures
– chemical fixing
• protects fine cellular substructure and morphology
of larger, more delicate organisms
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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
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Dyes and Simple Staining
• simple staining
– a single staining agent is used
– basic dyes are frequently used
• dyes with positive charges
• e.g., crystal violet
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Differential Staining
• divides microorganisms into groups based on
their staining properties
– e.g., Gram stain
– e.g., acid-fast stain
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Gram staining
• most widely used differential staining
procedure
• divides Bacteria into two groups based on
differences in cell wall structure
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primary
stain
mordant
decolorization
counterstain
positive
Figure 2.14 negative
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Figure 2.15c Escherichia coli – a gram-negative rod
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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
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Staining Specific Structures
• Negative staining
– often used to visualize capsules surrounding
bacteria
– capsules are colorless against a stained
background
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Staining Specific Structures
• Spore 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
Figure 2.20
35
The Transmission Electron Microscope
• 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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EM
Figure 2.23
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Specimen Preparation
• analogous 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
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Other preparation methods
• 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)
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Figure 2.25 Copyright © The McGraw-Hill Companies,
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Ebola
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Fly head
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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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Figure 2.27
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Newer Techniques in Microscopy
• confocal
microscopy and
scanning probe
microscopy
• have extremely
high resolution
• can be used to
observe individual
atoms
Figure 2.20
45
Confocal Microscopy
• confocal scanning laser microscope
• laser beam used to illuminate spots on
specimen
• computer compiles images created from each
point to generate a 3-dimensional image
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Figure 2.29
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Figure 2.30
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Scanning Probe Microscopy
• 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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Scanning Probe Microscopy
• 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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