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CHAPTER 2: Observing the Microbial Cell
CONCEPT MAP
I. Section 2.1: Observing Microbes
A. Resolution of objects
i. Resolution—smallest distance by which two objects can be separated
and still be distinguished
B. Resolution differs from detection
i. Detection—the ability to determine the presence of an object.
a. The eye can detect the presence of mold but can’t resolve individual
cells
ii. Magnification—increase in the apparent size of an object
C. Microbial size and shape
i. Prokaryotes are generally between 0.4–10 m
ii. Three common prokaryotic shapes
a. Bacilli—rod shaped
b. Cocci—spherical shaped
c. Spiral—corkscrew shaped
D. Microscopy for different size scales
i. Different types of microscopy are used to view structures of different sizes
II. Section 2.2: Optics and Properties of Light
A. Light carries information
i. Electromagnetic radiation interacts with objects and acquires information
that can be used to detect the objects
ii. Conditions necessary for electromagnetic radiation to resolve and object
a. Contrast between object and surroundings
b. Wavelength smaller than object
c. Detector with enough resolution for that wavelength
B. Light interacts with objects
i. Particles of light called photons interact with objects in many ways
a. Absorption—light energy is acquired by object
b. Reflection—wave front bounces off of object at angle equal to its
incident angle
c. Refraction—bending of light when it enters a substance that slows
its speed
d. Scattering—wave front interacts with object of smaller dimension
than the wavelength
C. Refraction enables magnification
i. Magnification requires refraction of light through medium of high refractive
index
D. Magnification and resolution
i. Empty magnification—magnification without increase in resolution
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III. Section 2.3: Bright-Field Microscopy
A. Increasing resolution
i. Resolution depends on
a. Wavelength of light
b. Contrast
c. Lens quality and magnifying power
d. Position of the focal plane
B. The compound microscope
i. Uses a system of lenses to achieve magnification and resolution
a. Condenser lens—concentrates light from light source to slide
b. Objective lenses—closest to specimen; typically magnify
10×, 40×, or 100×
c. Ocular lens—closest to eye; typically magnifies 10×
ii. Steps for observing specimen
a. Position specimen in middle of field of view
b. Optimize light—need more at higher powers
c. Focus objective lens
C. Is the object in focus?
i. Object is in focal plane of lens when edges of object appear sharp and distinct
D. Fixing and staining improve resolution and contrast
i. Wet mount—water and specimen on slide with cover slip
a. Can observe living cells in natural state
b. Most cells are transparent so show little contrast
ii. Fixation—cells are treated with alcohol or heat to make them adhere to slide
iii. Stains—adhere to bacteria to increase contrast
E. Different kinds of stains
i. Simple stain—uses one stain to color all cells
ii. Differential stain—distinguishes between different types of bacteria by using
different stains
a. Gram stain—bacteria are classified as Gram positive or Gram
negative based on whether they retain crystal violet stain
b. Acid-fast stain—carbolfuchsin is used to stain Mycobacterium species
c. Spore stain—malachite green and heat are used to stain endospores
d. Negative stain—stains background but not cells; used to view
capsules
e. Antibody stain—antibody proteins linked to fluorophores bind
specific components of cells
IV. Section 2.4: Dark-Field, Phase-Contrast, and Interference Microscopy
A. Dark-field microscopy
i. Uses scattered light so cells appear bright on dark background
ii. Can be used to view very small microbes and motility
iii. Disadvantage—particulates will scatter light; can be difficult to distinguish
from microbes from particulates
B. Phase-contrast microscopy
i. Enhances differences in refractive index so live cells can be observed without
staining
C. Interference microscopy
i. Superimposes interference bands on an image, accenting small differences
in refractive index
Full file at http://TestbanksCafe.eu/Test-Bank-for-Microbiology-An-Evolving-Science-2nd-EditionSlonczewski
V. Section 2.5: Fluorescence Microscopy
A. Fluorescence requires excitation and emission at different wavelengths
B. Fluorophores can label specific parts of cells by
i. Chemical affinity
ii. Labeled antibodies
iii. Gene fusion
iv. DNA hybridization
C. Laser confocal microscopy
i. Fluorescence is used along with laser optics to produce 3D images
VI. Section 2.6: Electron Microscopy
A. The electron microscope focuses beams of electrons and achieves resolution 1000×
resolution of light microscopy
i. Transmission electron microscopy
a. Electrons are transmitted through specimen
ii. Scanning electron microscopy
a. Electrons scan surface of specimen and are reflected to produce 3D
image
B. Electron microscopy requires specialized sample preparation
i. Specimen can be embedded in polymer and cut into thins sections using
microtome then coated with heavy metal
ii. Specimen can be sprayed onto copper grid then coated with heavy metal
iii. Specimen can be flash frozen for cryo-EM
C. Microscopy results require careful interpretation
i. Artifact—microscopic structure that is interpreted incorrectly
D. Emerging methods of microscopy
i. Cryo-EM
a. Samples are flash frozen in water solution; very high resolution
ii. Atomic force microscopy
a. Measure van der Waals forces between atoms on cell surface
and a sharp tip; very high resolution
VII. Section 2.7: Visualizing Molecules
A. X-ray diffraction
i. Uses X-ray interference patterns to generate computational model
of crystallized macromolecules
ii. Cryocrystallography—uses frozen crystals to determine structures
of macromolecular complexes