Chapter 2

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Chapter 2
The Study of Microbial

Structure:
Microscopy and Specimen
Preparation
1
Microscopy
• microorganisms range in size from
the smallest, viruses which are
measured in nanometers (nm), to the
largest, which are about 200
micrometers (µm).
2
Table 2.1
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 indices of
the two media forming the interface
4
Figure 2.1
5
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
6
Figure 2.2
7
The Light Microscope

• many varieties
– bright-field microscope
– dark-field microscope
– phase-contrast microscope
– fluorescence microscope
– confocal microscope
• are compound microscopes
– image formed by action of ≥2 lenses
8
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 lenses and the objective lenses
9
Figure 2.3
10
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
11
Figure 2.4
12
Figure 2.5
If air is replaced with immersion oil, many light rays that

did not enter the objective due to reflection and refraction
at the surfaces of the objective lens and slide will now do
so. This results in an increase in resolution and numerical
aperture.
13
Table 2.2
•working distance
— distance between the front surface of lens and
surface of cover glass or specimen when it is in sharp
focus
14
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
– 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
15
Figure 2.6
16
Figure 2.7
17
The Phase-Contrast Microscope

• converts slight differences in refractive index
and cell density into easily detected variations
in light intensity.
• some light rays from hollow cone of light
passing through an unstained cell are retarded
and out of phase and dark compared to the
bright background
• 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
18
Figure 2.9
19
Figure 2.10
20
Figure 2.8
21
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
– cell walls, endospores, granules, vacuoles,
and nuclei are clearly visible
22
Figure 2.11
23
The Fluorescence Microscope

• developed by O. Shimomuram, M. Chalfie,
and R. Tsien
• 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
• has applications in medical microbiology
and microbial ecology studies
24
Figure 2.12
25
Table 2.3
26
Fluorescence Microscopy
• essential tool in microbiology
– fluorochrome-labeled probes, such as
antibodies, or fluorochromes tag
specific cell constituents for
identification of unknown pathogens
– localization of specific proteins in cells
27
Figure 2.13
28
Figure 2.14
29
Preparation and Staining of

Specimens
• increases visibility of specimen
• accentuates specific morphological
features
• preserves specimens
30
Fixation
• preserves internal and external structures
and fixes them in position
• organisms usually killed and firmly
attached to microscope slide
– heat fixation – routine use with bacteria and
archaea
• preserves overall morphology but not internal
structures
– chemical fixation – used with larger, more
delicate organisms
• protects fine cellular substructure and morphology
31
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
32
Dyes and Simple Staining

• dyes
• 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 bacteria
33
Figure 2.17
34
Differential Staining
• divides microorganisms into groups
based on their staining properties
– e.g., Gram stain
– e.g., acid-fast stain
• differential stain used to detect
presence or absence of structures
– endospores, flagella, capsules
35
Gram Staining
• most widely used differential
staining procedure
• divides bacteria into two groups,
Gram positive and Gram negative,
based on differences in cell wall
structure
36
Figure 2.18
37
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 (mycolic
acid) is responsible for their staining
characteristics
38
Staining Specific Structures

• endospore staining
– heated, double staining technique
– bacterial endospore is one color and vegetative cell
is a different color
• capsule stain used to visualize capsules
surrounding bacteria
– negative stain - capsules may be colorless against a
stained background
• flagella staining
– mordant applied to increase thickness of flagella
39
Figure 2.19
40
Electron Microscopy
• electrons replace light as the
illuminating beam
• wavelength of electron beam is much
shorter than light, resulting in much
higher resolution
• allows for study of microbial
morphology in great detail
41
Figure 2.20
42
A Comparison of Light and Electron

Microscopy (Rhodospirillum rubrum)
43
The Transmission Electron

Microscope (TEM)
• electrons scatter when they pass
through thin sections of a specimen
• transmitted electrons are under
vacuum which reduces scatter and
are used to produce clear image
• denser regions in specimen, scatter
more electrons and appear darker
44
Figure 2.22
45
Figure 2.23
46
Table 2.4
47
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
materials, such as heavy metals, that
differentially scatter electrons
48
Other Preparation Methods

• negative stain
– heavy metals do not penetrate the specimen
but render dark background
– used for study of viruses, bacterial gas
vacuoles
• shadowing
– coating specimen with a thin film of a heavy
metal only on one side
– useful for viral morphology, flagella, DNA
49
Figure 2.24
50
Other Preparation Methods

• freeze-etching
– freeze specimen then fracture along
lines of greatest weakness (e.g.,
membranes)
– allows for 3-D observation of shapes of
intracellular structures
– reduces artifacts
51
Freeze-Etching (Thiobacillus kabobis)
52
The Scanning Electron

Microscope
• uses electrons reflected from the surface
of a specimen to create detailed image
• produces a realistic 3-dimensional image
of specimen’s surface features
• can determine actual in situ location of
microorganisms in ecological niches
53
Figure 2.26
54
Scanning Electron Micrograph of

Mycobacterium tuberculosis
55

Chapter 2

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Chapter 2

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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

The Study of Microbial

Lenses and the Bending of

The Light Microscope

The Bright-Field Microscope

If air is replaced with immersion oil, many light rays that

The Dark-Field Microscope

The Phase-Contrast Microscope

The Differential Interference

The Fluorescence Microscope

Preparation and Staining of

Dyes and Simple Staining

Dyes and Simple Staining

Staining Specific Structures

A Comparison of Light and Electron

The Transmission Electron

Other Preparation Methods

Other Preparation Methods

Freeze-Etching (Thiobacillus kabobis)

The Scanning Electron

Scanning Electron Micrograph of

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