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Microscopy
Chapter 4
Microscopy, Staining and Classification
• General Principles of Microscopy
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–
–
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Wavelength of radiation – uses light or electrons
Magnification – increase in size of object
Resolution - clarity
Contrast – difference between 2 objects or object
and background; stain to increase contrast
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Figure 4.1 The electromagnetic spectrum
400 nm
700 nm
Visible light
Gamma rays
10–12m
X UV
rays light
10–8m
Infra- Microred
wave
Radio waves and
Television
Increasing wavelength
10–4m
100m
103m
Crest One wavelength
Trough
Increasing resolving power
Figure 4.2 Light refraction and image magnification by a convex glass lens-overview
Light
Glass
Air
Focal point
Specimen
Convex
lens
Inverted,
reversed, and
enlarged
image
Figure 4.3 The limits of resolution of the human eye and of various types of microscopes
Diameter
of DNA Ribosomes
Proteins Viruses
Atoms
Amino
acids
Typical bacteria
and archaea
Flea
Chloroplasts
Mitochondrion
Large
protozoan
(Euglena)
Chicken
egg
Human red
blood cell
Scanning tunneling microscope
(STM) 0.01 nm–10 nm
Transmission electron microscope (TEM)
0.078 nm–100 µm
Scanning electron microscope (SEM)
0.4 nm–1 mm
Atomic force
microscope (AFM)
1 nm–10 nm
Compound light microscope (LM)
200 nm–10 mm
Unaided human eye
200 µm–
Microscopy
• Light Microscopy
– Bright-field microscopes – uses light
– 1.
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Simple
Contain a single magnifying lens
Similar to magnifying glass
Leeuwenhoek used simple microscope to
observe microorganisms
Microscopy
– 2. Compound
– Series of lenses for magnification
– Light passes through specimen into
objective lens
– Have one or two ocular lenses
– Total magnification (objective lens X ocular
lens – 10X)
– Low – red – 4X – total 40X
– medium - yellow – 10X – total100X
– high – blue – 40X – total 400X
– Oil immersion – white -100x – total
1000X - Oil immersion lens increases
resolution
- Most have condenser lens (direct light
through specimen)
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Microscopy
- 3. Darkfield microscope – background dark –
used for unstained organisms
- 4. Fluorescent microscope – specimen stained
with fluorescent dyes
- 5. Phase-contrast microscope – allows
observation of dense structures in living
organisms – no staining needed – sharp
contrast
Figure 4.4 A bright-field, compound light microscope-overview
Ocular lens
Line of vision
Remagnifies the image formed by
the objective lens
Body
Transmits the image from the
objective lens to the ocular lens
using prisms
Arm
Objective lenses
Primary lenses that
magnify the specimen
Stage
Holds the microscope
slide in position
Condenser
Focuses light
through specimen
Diaphragm
Controls the amount of
light entering the condenser
Illuminator
Light source
Coarse focusing knob
Moves the stage up and
down to focus the image
Fine focusing knob
Base
Ocular lens
Path of light
Prism
Body
Objective
lenses
Specimen
Condenser
lenses
Illuminator
Figure 4.5 The effect of immersion oil on resolution-overview
Microscope
objective
Refracted light
rays lost to lens
Microscope
objective
Lenses
More light
enters lens
Glass cover slip
Glass cover slip
Slide
Slide
Specimen
Light source
Without immersion oil
Immersion oil
Light source
With immersion oil
Microscopy
• Electron Microscopy
– Light microscopes cannot resolve structures
closer than 200 nm
– Greater resolving power and magnification
– Magnifies objects 10,000X to 100,000X
– Detailed view of bacteria, viruses, ultrastructure,
and large atoms
– Two types
– Transmission electron microscopes
– Scanning electron microscopes
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Figure 4.11 A transmission electron microscope (TEM) -overview
Light microscope
(upside down)
Column of transmission
electron microscope
Lamp
Electron gun
Condenser
lens
Condenser lens
(magnet)
Specimen
Specimen
Objective lens
Objective lens
(magnet)
Eyepiece
Projector lens
(magnet)
Final image
seen by eye
Final image on
fluorescent screen
Figure 4.12 Scanning electron microscope (SEM)
Electron gun
Magnetic
lenses
Primary
electrons
Beam
deflector coil
Scanning
circuit
Secondary
electrons
Specimen
Specimen
holder
Vacuum
system
Photomultiplier
Detector
Monitor
Figure 4.13 SEM images-overview
Microscopy
• Probe Microscopy
– Magnifies more than 100,000,000X
– Two types
– Scanning tunneling microscopes
– Atomic force microscopes
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Figure 4.14 Probe microscopy-overview
DNA Enzyme
Staining
• Principles of Staining
– Staining increases contrast and resolution by
coloring specimens with stains/dyes
– Smear of microorganisms (thin film) made prior
to staining
– Acidic dyes stain alkaline (base) structures
– Nigrosin, India Ink
– Basic dyes stain acidic structures
– Crystal violet, methylene blue, safranin,
malachite green
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Figure 4.15 Preparing a specimen for staining
Spread culture in
thin film over slide
Air dry
Pass slide through
flame to fix it
Staining
• Simple Stains – single basic dye
• Differential Stains – use more than 1 dye
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–
–
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Gram stain – for positive or negative
Acid-fast stain – for waxy cell walls
Endospore stain – uses heat to force stain
Histological stain – for tissue (cancer or fungi)
• Special Stains
– Negative (capsule) stain – stains background
– Flagellar stain
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Figure 4.16 Simple stains-overview
Figure 4.17 The Gram staining procedure-overview
Slide is flooded with crystal
violet for 1 min, then rinsed
with water.
Slide is flooded with iodine
for 1 min, then rinsed with
water.
Result: All cells are stained
purple.
Result: Iodine acts as a
mordant; all cells remain
purple.
Slide is flooded with solution
of ethanol and acetone for
10–30 sec, then rinsed with
water.
Slide is flooded with safranin
for 1 min, then rinsed with
water and blotted dry.
Result: Smear is decolorized;
Gram-positive cells remain
purple, but Gram-negative
cells are now colorless.
Result: Gram-positive cells
remain purple, Gram-negative
cells are pink.
Figure 4.18 The Ziehl-Neelsen acid-fast stain
Figure 4.19 Schaeffer-Fulton endospore stain of Bacillus anthracis
Figure 4.20 Negative (capsule) stain of Klebsiella pneumoniae
Bacterium
Capsule
Background
stain
Figure 4.21 Flagellar stain of Proteus vulgaris
Flagella
Classification and Identification of Microorganisms
– Taxonomy consists of classification,
nomenclature, and identification
– Organize large amounts of information
about organisms
– Make predictions based on knowledge of
similar organisms
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Classification and Identification of Microorganisms
• Linnaeus and Taxonomic Categories
– Linnaeus
– Classified organisms based on characteristics
in common
– Organisms that can successfully interbreed
called species
– Used binomial nomenclature in his system –
genus and species
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Classification and Identification of Microorganisms
• Linnaeus and Taxonomic Categories
– Linnaeus proposed only two kingdoms
– Later taxonomic approach based on five
kingdoms
– Animalia, Plantae, Fungi, Protista, and
Prokaryotae
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Classification and Identification of Microorganisms
• Linnaeus and Taxonomic Categories
– Linnaeus’s goal was to classify organisms to
catalogue them
– Modern goal is to understand relationships
among groups of organisms
– Reflect phylogenetic hierarchy – grouping
organisms reflecting their evolution from
common ancestors
– Emphasis on comparison of organisms’
genetic material
– Led to proposal to add domain
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Classification and Identification of Microorganisms
• Domains
– Proposal of three domains as determined by
ribosomal nucleotide sequences
– Eukarya, Bacteria, and Archaea
– Cells in the three domains differ by other
characteristics
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Classification and Identification of Microorganisms
• Taxonomic and Identifying Characteristics
– Physical characteristics
– Biochemical tests – microbes ability to utilize or
produce certain chemicals
– Serological tests – use antiserum (serum
containing antibodies); clumping of antigen with
antibodies (agglutination) indicates presence of
targeted cells
– Phage typing – reveals if 1 bacterial strain is/is
not susceptible to a particular phage – plaques
(clear area) form where bacteria killed by phage
– Analysis of nucleic acids
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Figure 4.23 Two biochemical tests for identifying bacteria-overview
Gas bubble
Acid with gas
Inverted tubes to trap gas
Acid with no gas
Inert
Hydrogen
sulfide
produced
No
hydrogen
sulfide
Figure 4.24 One tool for the rapid identification of bacteria, the automated MicroScan system
Wells
Figure 4.25 An agglutination test, one type of serological test-overview
Negative result
Positive result
Negative result
Positive result
Figure 4.26 Phage typing
Bacterial lawn
Plaques
Classification and Identification of Microorganisms
• Taxonomic Keys
– Dichotomous keys
– Series of paired statements where only one of two
“either/or” choices applies to any particular
organism
– Key directs user to another pair of statements, or
provides name of organism
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Figure 4.27 Use of a dichotomous taxonomic key-overview
Gram-positive
cells?
No
Yes
Gram-positive
bacteria
Rod-shaped
cells?
No
Yes
Can
tolerate
oxygen?
Cocci and
pleomorphic
bacteria
No
Yes
Ferments
lactose?
Obligate
anaerobes
No
Yes
Non-lactosefermenters
Can use citric
acid (citrate)
as sole carbon
source?
No
Yes
Produces gas
from glucose?
No
Shigella
Produces hydrogen
sulfide gas?
No
Yes
Escherichia
Yes
Produces
acetoin?
No
Citrobacter
Salmonella
Yes
Enterobacter