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PowerPoint® Lecture
Presentations prepared by
Mindy Miller-Kittrell,
North Carolina State
University
C H A P T E R
© 2017 Pearson Education, Inc.
Microscopy,
Staining, and
Classification
4
CSLO1. Describe distinctive characteristics and diverse
growth requirements of prokaryotic organisms compared
to eukaryotic organisms.
PSLO3. Will demonstrate proficiency and safe practice
sin the use of laboratory equipment and basic
laboratory techniques.
CSLO CHECK
© 2017 Pearson Education, Inc.
Principles behind Microscopy
Types of Microscopes
Light Microscopes
Electron Microscopes
Different staining methods for Microscopy
CH4-section1
Probe Microscopes
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Figure 4.3 The limits of resolution (and some representative objects within those ranges) of the human eye and of
various types of microscopes.
Electron Microscopes
Probes Microscopes
Light Microscopes
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3 General Principles of Microscopy which
influence observation of microbes
• Magnification
• Resolution
• Contrast
Official definitions
Magnification, the ratio of an object’s image
size to its real size
Resolution, the measure of the clarity of the image, or the
minimum distance of two distinguishable points
Contrast, visible differences in brightness between parts of
the sample
FACT 4.1CONCEPT 4.1
© 2017 Pearson Education, Inc.
3 General Principles of Microscopy
• A) Magnification (enlarge)
• B) Resolution (tell apart 2 objects close
together)
• C) Contrast (Differences in intensity between
two objects)
How to increase all 3 of these
to achieve optimal observation of
microbes?
FACT 4.1CONCEPT 4.1
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A) Magnification vs B) Resolution
High Magnification but Low Resolution
Need both high Resolution
and Magnification
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VIBGYOR
Smaller the object = Use smaller
wavelength
To increase A) Magnification and B) Resolution
Use smaller wavelength CONCEPT 4.2
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Light vs Electron microscope
Visible Light
Electron
has very
small
wavelength
compared
to visible
light
Which microscope will let you see
smaller things ?
CONCEPT 4.2
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Light vs Electron microscope
Uses visible light Uses electron
Magnifies objects 100,000XMagnifies objects 1000X
FACT 4.2
glass lenses magnet
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Light Microscopy
• In a light microscope (LM), visible light is passed
through a specimen and then through glass
lenses
• Glass lenses focuses light and enlarges and
resolves objects
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Light
Air
Glass
Light changes direction (refracts) when
it moves from one medium to another
Air
CONCEPT 4.3
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Focal point
Specimen Convexlens
Inverted,
reversed, and
Enlarged
image
5 50
Magnification = 50/5 = 10X
Magnify using lenses
polish glass
CONCEPT 4.4
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• Light Microscopy
• Bright-field microscopes
• Simple Microscopes
• Contain a single magnifying lens
• Similar to magnifying glass
• Leeuwenhoek used simple microscope to observe
microorganisms for the first time in history
Fresh pond water
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Light Microscopy (Bright-field microscopes)
• Compound – multiple lenses
• Series of lenses for magnification
• Light passes through specimen into
objective lens – 10X, 40X, 100X
• Have one or two ocular lenses 10X
Total magnification
= magnification of objective lens
X magnification of ocular lens
e.g. 10 times X 10 times
= 100 times
FACT 4.3CONCEPT 4.5
10X
40X
100X
10X
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Light
Air
Glass
Light changes direction (refracts) when
it moves from one medium to another
Air
CONCEPT 4.3
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Figure 4.5 The effect of immersion oil on resolution.
Glass cover slip
Slide
Specimen Light source
Without immersion oil
Lenses
Immersion oilGlass cover slip
Slide
Light source
With immersion oil
Microscope
objective
Refracted light
rays lost to lens
Microscope
objective
More light
enters lens
immersion oil
Increases magnification
and resolution
FACT 4.4
Oil immersion lens (100X) is a
special type of objective lensimmersion oil reduces
scattering of light
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Brightfield(stained specimen)
50
µm
Staining increases contrast
Brightfield(unstained specimen)
FACT 4.5
Staining kills cell
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Different types of Microscopy
Light Microscopy
1) Bright-field microscopes show bright background
FACT 4.6
Four kinds of light microscopy
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Bacterium
Bright field Dark field
2) Dark-field microscopy
• Specimen appears light against dark background
• Increases contrast and enables observation of more
details
• Examine live specimens
1) 2)
DarkBright-field microscopes show dark background
FACT 4.7
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Phase contrast Nomarski
3A) Phase-contrast microscopy
3B) Differential interference contrast microscope aka Nomarski
3) Phase Microscopy – 2 types
Used to examine living organisms or specimens that would
be damaged/altered by attaching them to slides or staining
Light rays in phase produce brighter image, while light rays out
of phase produce darker image.
FACT 4.8
Contrast is created by combining light waves in or out of
phase
Shows 3D
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4) Fluorescent microscopes
• Direct UV light source at specimen
• Fluorescent stains absorb UV and radiates energy back
as a longer, visible wavelength
UV light increases resolution and contrast
UV
FACT 4.9
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4A) Fluorescence microscopy – 3 types
Can help you look at object of interest only
FACT 4.10
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4B) Immunofluorescence
Antibodies
Bacterium
Cell-surfaceantigens
Bacterial cell withbound antibodiescarrying dye
Fluorescent dye
Antibodiescarrying dye
help you detect specific pathogens
by attaching fluorescent dyes to the antibody
specific for that pathogen
FACT 4.11
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4C) Confocal microscopes
Use UV lasers to illuminate fluorescent chemicals in a single plane
Computer constructs 3-D image from digitized images
FACT 4.12
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Microscopy
• Electron Microscopy
• Light microscopes cannot resolve structures closer than 200 nm
• Electron microscopes have greater resolving power and
magnification than light microscopy
• Magnifies objects 10,000X to 100,000X
• Detailed views of bacteria, viruses, internal cellular structures,
molecules, and large atoms
2 types of electron microscopes
• Transmission electron microscopes
• Scanning electron microscopes
FACT 4.13
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Figure 4.11 A transmission electron microscope (TEM)
Lamp
Light microscope(upside down)
Column of transmissionelectron microscope
Lamp
Condenser
lens
Specimen
Objective lens
Eyepiece
Final image
seen by eye
Electron gun
Specimen
Objective lens
(magnet)
Projector lens
(magnet)
Final image on
fluorescent screen
All electron microscopy needs vacuum - Why ?
TEM needs sections - why ?
FACT 4.13
TEM sees transmitted electrons
Consequences = Only dead objects seen
CONCEPT ?
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Figure 4.12 Scanning electron microscope (SEM).
SEM sees scattered electrons
SEM sees surface of microbes
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• Probe Microscopy – 2 types
• Probe travels over specimen
Scanning tunneling
microscopes
(highest magnification
and resolution)
Atomic force
microscopes
FACT 4.14
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Table 4.2 Comparison of Types of Microscopes (1 of 3)
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Table 4.2 Comparison of Types of Microscopes (2 of 3)
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Table 4.2 Comparison of Types of Microscopes (3 of 3)
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Staining for Light Microscopy
• Principles of Staining
• Simple Staining = one stain/dye
• Differential Staining = more than one stain – to
differentiate between different types of microbes
• Basic dyes (positively charged) stain acidic (negatively
charged) structures
• Acidic dyes (negatively charged) stain alkaline
structures
++
+ +++++++ +
++ ----
--+++ +
++ --- --
++
+ ++++
Basic dyes
CONCEPT 4.6
FACT 4.15
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Inside of most bacterial cells is
negatively charged
- --- --++
+ ++++
Basic dyes
Positive Staining
----- -
Therefore Basic dyes are more commonly used
to stain bacterial cells
CONCEPT 4.6
© 2017 Pearson Education, Inc.
Inside of most bacterial cells is
negatively charged
- --- ---
Acidic dyes
----- -
Therefore Acidic dyes
are more commonly used to stain background
---
--
- -
-- --
Negative Staining
CONCEPT 4.6
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Gram Staining MechanismFACT 4.16
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Gram Staining Method
Crystal Violet
Primary Stain Step
Gram positive cell wall
Gram negative cell wall
FACT 4.16
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Gram Staining Method
Gram’s Iodine
Mordant Step
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Gram Staining Method
Alcohol
Decolorizing Step
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Gram Staining Method
Safranin
Counter Stain Step
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Table 4.3 Some Stains Used for Light Microscopy
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Staining for Electron Microscopy
Chemicals
containing heavy
metals
are used as stains
for electron
microscopy
FACT 4.17
CONCEPT 4.7
transmission electron microscopy
© 2017 Pearson Education, Inc.
Other Identifying
Characteristics
Physical characteristics
Biochemical tests
Serological tests
Phage typing
Analysis of nucleic acids
How to classify microbes
Different Methods to identify microbes
Staining MethodsHow to name microbes
Taxonomy
1
2
3
CH4-section2
© 2017 Pearson Education, Inc.
• Microbe Identifying Characteristics
• Physical characteristics
• Biochemical tests
• Serological tests
• Phage typing
• Analysis of nucleic acids
© 2017 Pearson Education, Inc.
Physical characteristics
Protozoa, fungi, algae, and parasitic worms can often be
identified based only on their morphology
Some bacterial
colonies have distinct
appearance used for
identification
CONCEPT 4.8
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No
hydrogen
sulfide
Hydrogen
sulfide
producedAcid with gas Acid with no gas Inert
Gas bubble Inverted tubes to trap gas
Biochemical tests
CONCEPT 4.9
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An agglutination test
Serological (Blood) Tests CONCEPT 4.10
Antibody-Antigen
Complex causes
agglutination
ZIKA virus Ebola virus
Patient Blood
(contains antibodies)
plus add virus on slide
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Bacterial lawn
Plaques
(holes)
Phage typing
Identifying bacteria based on which
phage infects and lyses the bacteria
CONCEPT 4.11
Phage T4 Phage T7
Bacteria A Plaques No
Plaques
Bacteria B No
Plaques
Plaques
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Analysis of nucleic acids
Nucleic acid sequence can be used to classify and identify
microbes
Prokaryotic taxonomy now includes the G + C content of
an organism's DNA
CONCEPT 4.12
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• 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
CONCEPT 4.13
© 2017 Pearson Education, Inc.
Other Identifying
Characteristics
Physical characteristics
Biochemical tests
Serological tests
Phage typing
Analysis of nucleic acids
How to classify microbes
Different Methods to identify microbes
Staining MethodsHow to name microbes
Taxonomy
1
2
3
© 2017 Pearson Education, Inc.
humans belong to the genus Homo
species Homo sapiens.
naming species of living things by giving
each species a name composed of two parts
noun-genus
Capitalizedadjective-specific epithet
Binomial nomenclature
Homo sapiens
CONCEPT 4.14
Each species has a unique name
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Homo naledi (2015)
Homo sapiens
members of Genus are very different
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Genus specific epithet
Genus specific epithet
X
XGenus specific epithetYGenus specific epithetY
Escherichia coliEscherichia coliE. coli
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Figure 3.12 Bacterial shapes and arrangements.
2) strepto
1) coccus 2) bacillus
3 major shapes/morphology
3) spirillum
2 major arrangements
1) staphylo
FACT 4.18
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Microbe Naming Rules
Genus is based on shape and arrangement
specific epithet is based on unique characteristic
CONCEPT 4.14
(Arrangement+shape) (unique characteristic)
(specific epithet)(Genus)
Streptobacillus pnemoniae
Escherichia coli
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cocci bacilli
arrangement
shape
Streptococci
Staphylococci
Streptobacilli
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Staphylococcus partyanimalia
Lets name this microbe
Genus specific epithet
© 2017 Pearson Education, Inc.
Other Identifying
Characteristics
Physical characteristics
Biochemical tests
Serological tests
Phage typing
Analysis of nucleic acids
How to classify microbes
Different Methods to identify microbes
Staining MethodsHow to name microbes
Taxonomy
1
2
3
consists of
Identification,
Nomenclature and
classification
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Taxonomy consists of identification, nomenclature, and
classification
Why do Taxonomy ?
• Organize large amounts of information about organisms
• Make predictions based on knowledge of similar
organisms
CONCEPT 4.15
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His system
classified organisms
based on
characteristics in
common
Grouped organisms
that can
successfully
interbreed into
categories called
species
Used binomial
nomenclature
FACT 4.19Carolus Linnaeus and Taxonomic Categories
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Domains are highest taxonomic units
• Carl Woese compared nucleotide sequences of rRNA
subunits
• Proposal of three domains as determined by ribosomal
nucleotide sequences – genetic information
• Eukarya, Bacteria, and Archaea
Goal of modern taxonomy
is to reflect phylogenetic hierarchy
– not just morphology but
understanding genetic relationships
among organisms – build
phylogenetic or evolutionary trees
Bat vs Bird
CONCEPT 4.16
FACT 4.20