Microbiology An Evolving Science Third Edition Joan L.
Slonczewski and John W. Foster Copyright 2014 W. W. Norton &
Company, Inc. Permission required for reproduction or display
PowerPoint Lecture OutlinesPrepared by Johnny El-Rady, University
of South Florida 2 Observing the Microbial Cell 2 Chapter Overview
How microorganisms are observed The physics of light The
bright-field microscope Staining bacterial cells The fluorescence,
dark-field, and phase-contrast microscopes The electron microscope
Cutting-edge microscopes How molecules are visualized 3 Figure 1.13
4 Introduction Since Leeuwenhoeks time, powerful microscopes have
been devised to search for microbes in unexpected habitats - e.g.,
the human stomach Microscopy revealedthe presence ofHelicobacter
pylori,the cause ofstomach ulcers Figure 2.1 5 Microbes differ in
size, over a range of a few orders of magnitude, or powers of ten.
- Eukaryotic microbes - Protozoa, algae, fungi - 10100 mm -
Prokaryotes - Bacteria, archaea - 0.410 mm Nevertheless, a few
bacterial species are large enough to be seen by the unaided eye -
Thiomargarita namibiensis Microbial Size 6 Figure 2.4 7 Prokaryotic
cell structures are generally simpler than those of eukaryotes
Certain shapes of bacteria are common to many taxonomic groups -
Bacilli = rods - Cocci = spheres - Spiral forms - Spirochetes -
Spirilla Microbial Shape 8 Figure 2.6 9 2.1 Observing Microbes The
size at which objects become visible depends on the resolution of
the observers eye Resolution is the smallest distance by which two
objects can be separated and still be distinguished The resolution
of the human retina is about150 mm (1/7 mm) Contrast is the ability
to distinguish an object from its surrounding (background) 10 We
define what is visible and what is microscopic in terms of the
human eye Figure 2.2 11 Different microscopes are required to
resolve various cells and subcellular structures. Microscopy for
Different-Sized Scales Figure 2.7 12 Detection is the ability to
determine the presence of an object Resolution Is Different from
Detection Figure 2.3 Magnification means an increase in the
apparent size of an image to resolve smaller separations between
objects 13 Theoreticalresolution limit = /2 14 Absorption means
that the photons energy is acquired by the absorbing object
Reflection means that the wavefront bounces off the surface of an
object Refraction is the bending of light as it enters a substance
that slows its speed Scattering occurs when the wavefront interacts
with an object smaller than the wavelength of light Light Interacts
with an Object 15 Figure 2.9 16 2.2 Optics and Properties of Light
Light is part of the spectrum of electromagnetic radiation.-
Wavelength of visible light = 400750 nm For electromagnetic
radiation to resolve an object, certain conditions must exist: 1.
Contrast between object and its medium 2. Wavelength smaller than
the object 3. Magnification 17 Magnification requires the bending
of light rays, as in refraction Wavefronts of lightshift direction
as theyenter a substance ofhigher refractive index Magnification by
a Lens Figure 2.10 18 When light rays enter a parabolic lens,
parallel rays each bend at an angle such that all of the rays meet
at a certain point, called the focal point Figure 2.11 19 Figure
2.12 20 Figure 2.14 Figure 2.15 R = /2*NA 21 It is resolution, not
magnification, that limits the ability of what we can see with a
microscope - Indeed, magnification without increasing detail is
called empty magnification The resolution of detail in microscopy
is limited by the wave nature of light- Light rays actually form
wavefronts, which undergo interference - Can be constructive or
destructive Resolution of Detail 22 2.3 Bright-Field Microscopy
Generates a dark image of an object over a light background To
increase resolution: - Use shorter wavelength light - Lessen
contrast - Use immersion oil - Use wider lens closer to specimen -
Higher numerical aperture (NA) 23 A compound microscope is a system
of multiple lenses designed to correct or compensate for aberration
- Ocular lens - Objective lens Total magnification = magnification
of ocular multiplied by that of the objective The Compound
Microscope 24 Figure 2.16 -Objectives may be parfocal25 A simple
way to observe microbes is to place them in a drop of water on a
slide with a coverslip. - This is called a wet mount preparation. -
Advantages: - Observation of cells in natural state -
Disadvantages: - Little contrast between cell and background -
Sample may dry out quickly Preparing a Specimen for Microscopy 26
Figure 2.17 27 The detection and resolution of cells under a
microscope are enhanced by: - Fixation = cells are made to adhere
to a slide in a fixed position - Staining = cells are given a
distinct color- Most stains have conjugated double bonds or
aromatic rings, and one or more positive charges Fixation and
Staining 28 A simple stain adds dark color specifically to cells,
but not to the external medium or surrounding tissue - Most
commonly used stain is methylene blue A differential stain stains
one kind of cell but not another- The most famous differential
stain is the Gram stain Different Kinds of Stains 29 Figure 2.20 30
The Gram stain was devised in 1884 by the Dutch physician Hans
Christian Gram (18531938) - It differentiates between two types of
bacteria: - Gram-positive bacteria retain the crystal violet stain
because of their thicker cell wall - Gram-negative bacteria do not
Figure 2.21 31 Figure 2.22 32 Acid-fast stain = carbolfuchsin used
to stain Mycobacterium species Spore stain = malachite green used
to detect spores of Bacillus and Clostridium Negative stain =
colors the background, which makes capsules more visible Other
Differential Stains Figure 2.24 33 2.4 Fluorescence Microscopy In
fluorescence microscopy, the specimen absorbs light of a defined
wavelength, and then emits light of lower energy, thus longer
wavelength; that is, the specimen fluoresces Used to view marine
and pathogenic bacteria Figure 2.25 34 Excitation and Emission The
specimen absorbs light of a specific wavelength (the excitation
wavelength), then emits light at a longer wavelength (the emission
wavelength) Figure 2.26 35 The optical system for fluorescence
microscopy uses color filters - To limit incidentlight to
thewavelength ofexcitation andemitted light tothe wavelengthof
emission Figure 2.27 36 A fluorophore is a fluorescent chemical
compound - Its cell specificity can be determined in three ways: -
Chemical affinity - Labeled antibodies - DNA hybridization
Fluorophores for Labeling Figure 2.29 37 2.5 Dark-Field Microscopy
Dark-field optics enables microbes to be visualized as halos of
bright light against darkness Light shines at oblique angle - Only
light scattered by sample reaches objective - Makes visible objects
below resolution limit - Flagella - Very thin bacteria 38 Figure
2.33 Figure 2.32 Figure 2.31 39 2.5 Phase-Contrast Microscopy
Superimposes refracted light and transmitted light shifted out of
phase- Reveals differences in refractive index as patterns of light
and dark - Can be used to view live cells and cellular organelles
Figure 2.34 40 Figure 2.35 41 2.6 Electron Microscopy Electrons
behave like light waves - Very high frequency - Allow very great
resolution - A few nanometers Sample must absorb electrons - Coated
with heavy metal Electron beam and sample are in a vacuum - Lenses
are magnetic fields 42 Figure 2.36 43 2.6 Electron Microscopy Two
major types - Transmission electron microscopy (TEM) - Electrons
pass through the specimen - Reveals internal structures - Scanning
electron microscopy (SEM) - Electrons scan the specimen surface -
Reveals external features in 3D 44 Figure 2.37 The TEM closely
parallels the design of the bright-field microscope 45 The SEM is
arranged somewhat differently from the TEM Figure 2.38 46 The
specimens for electron microscopy can be prepared in several ways -
Embedded in a polymer for thin sections - Microtome is used to cut
slices - Sprayed onto a copper grid The specimen is then treated
with a heavy-metal salt such as uranyl acetate Note: For SEM,
specimen is coated with heavy metal and it is not sliced Sample
Preparation 47 Figure 2.39 Figure 2.40 48 2.6 Tomography In
cryo-EM, or electron cryomicroscopy, the specimen is flash-frozen -
Suspended in water and frozen rapidly in a refrigerant
Cryo-electron tomography, or electron cryotomography, avoids the
need to physically slice the sample for thin-section TEM - The
images are combined digitally to visualize the entire object in 3D
- Generates high-resolution models of virus particles 49 Figure
2.42 50 Figure 2.43 51 Figure 2.44 52 Scanning probe microscopy
(SPM) enables nanoscale observation of cell surfaces The atomic
force microscope (AFM) is an example of an SPM It measures the van
der Waals forces between electron shells of adjacent atoms of the
cell surface and the sharp tip It can be used to observe live
bacteria in water or exposed to air (unlike electron microscopy)
Emerging Methods of Microscopy 53 Figure 2.45 54 2.7 Visualizing
Molecules X-ray diffraction analysis - For samples that can be
crystallized, X-ray diffraction makes it possible to fix the
position of individual atoms in a molecule - A beam of X-rays is
shot at a crystallized sample - Many molecules in identical
conformation - X-rays diffract according to position of atoms -
Compute position of atoms from pattern of scattered X-rays 55
Figure 2.46 56 Today, X-ray data undergo digital analysis to
generate sophisticated molecular models - Example: the anthrax
lethal factor - A toxin producedby Bacillus anthracis - Note: The
model wasencoded in a proteindata bank (PDB)text file Figure 2.48
57 Chapter Summary When observing microbes, resolution and
magnification are paramount Different kinds of microscopes are
required to resolve cells and subcellular structures: -
Bright-field: employs various stains - Fluorescence: employs
fluorophores for labeling - Dark-field: detects unresolved objects
- Phase-contrast: exploits differences in refractive indices 58
Chapter Summary Electron microscopes use beam of electrons instead
of light rays - TEM: provides internal details in 2D - SEM:
provides external details in 3D Scanning probe microscopes (SPMs)
include the atomic force microscope (AFM) - Allow observation of
living cells in water or in air Molecules can be visualized by
X-ray crystallography 59 Concept CheckSection 2.1 Which of the
following statements about the size of microbes is FALSE? a)
Eukaryotic microbes tend to have a size of10100 mm. b) Prokaryotic
microbes tend to have a size that is less than 10 mm. c) A few
bacterial species are large enough to be seen by the unaided eye.
d) Choose this answer if all the above are true. 60 Concept
CheckSection 2.2 Which of the following properties is most
important for a lens to magnify an image? a)Absorption b)Reflection
c)Scattering d)Refraction 61 Concept CheckSection 2.3 You are
observing a bacterium using a 10 ocular lens and a 45 objective
lens. What would the total magnification be? a) 10 b) 45 c)145
d)450 e)Need more information 62 Concept CheckSection 2.3 What is
the correct order of reagents in the Gram stain? a) Iodine, crystal
violet, ethanol, safranin b) Crystal violet, iodine, ethanol,
safranin c)Crystal violet, ethanol, iodine, safranin d) Iodine,
ethanol, safranin, crystal violet e)Safranin, ethanol, iodine,
crystal violet63 Concept CheckSection 2.4 In fluorescence
microscopy, the specimen absorbs incident light and then re-emits
it at a _______ energy and thus, a _______ wavelength. a)lower;
shorter b)lower; longer c)higher; shorter d)higher; longer 64
Concept CheckSection 2.5 Which of the following microscopes allows
the best view of bacterial flagella during motility? a)Bright-field
microscope b)Dark-field microscope c)Fluorescence microscope
d)Transmission electron microscope e) Scanning electron microscope
65 Concept CheckSection 2.5 Which of the following statements
aboutphase-contrast microscopy is true? a)It exploits differences
in refractive indices between cell parts and surrounding media.
b)It can be used to view live cells. c)Both A and B d)Neither A nor
B 66 Concept CheckSection 2.6 All of the following statements apply
to scanning electron microscopy EXCEPT a)The specimen is usually
fixed and embedded b)The embedded specimen is cut into thin
sections with a microtome c)It cannot be used to view live
specimens d)It provides 3D images of the specimen 67 Concept
CheckSection 2.6 Which of the following statements about theatomic
force microscope is true? a)It is an example of a scanning-probe
microscope b)It measures van der Waals forces c)Both A and B
d)Neither A nor B 68 Concept CheckSection 2.7 What is the best
technique for examining the presence of a chemical structure with a
diameter of 3 nm? a)Light microscopy b)Electron microscopy
c)Ultracentrifugation d)Tomography e)X-ray crystallography