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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

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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
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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
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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
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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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