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Microscopy
How to see the little ones
How small are they?
•  Viruses - 50-100 nanometers
•  Bacteria - 1-5 micrometers
•  Eukaryotic cells - 10-100 micrometers
Two major aspects of microscopy:
Magnification and Resolution
•  Magnification - an ability to make objects
appear larger, expressed as a number
combined with X (times). •  Example: A microscope magnifies a
specimen by 100X. (This means the
object looks 100 times as large as it really
is).
Two major aspects of microscopy:
Magnification and Resolution
•  Resolution - the ability to show detail, to see
magnified objects clearly.
•  Officially defined as the ability of an optical
system to distinguish between two adjacent
points.
•  Resolving power has to do with the
wavelength of light employed for viewing, as
well as other technical considerations.
Two major aspects of microscopy:
Magnification and Resolution
•  A good illustration of the relationship:
“Even if a light
microscope could be designed to magnify
e.g. 10,000X, its resolving power COULD
NOT be increased (due to the wavelength of
visible light), and the enlarged image would
be big but fuzzy”.
The Compound Light Microscope
(“Brightfield Microscopy”)
•  Two lenses: the ocular
(10x) and the objectives
(often 4x, 10x, 40x, and
100x)
•  Uses visible light
•  Maximum resolving
power = 200 nanometers;
maximum magnification
= 2000X
Cont'd
•  Specimens are
usually stained since
most microorganisms
are clear; this
increases the
refractive indices of
the specimen and the
medium.
Immersion oil
•  Immersion oil must be
used under high
magnification to
reduce loss of light;
the oil has a refractive
index similar to the
microscope slide.
•  Much better resolution
Other types of microscopy
•  Darkfield microscopy - specimen appears
light against a dark background (no
staining necessary)
•  Fluorescence microscopy - specimens are
stained with "fluorochromes", which
absorb and reradiate UV light. The sample
appears bright against a dark background.
(Often used for diagnostic purposes with
fluorescent antibodies)
Electron microscopy
•  A beam of electrons is used instead
of visible light
•  This allows much greater
magnification and resolution
•  Electromagnets act as lenses to
focus the beam
Two types
•  Transmission electron microscopy
•  Beam penetrates sample just like in light
microscopy. •  Samples must be very thin!
•  Highest magnification and resolution (up to
1,000,000x, 1 nM)
•  Similar in idea to brightfield microscopy
Scanning electron microscopy
•  Electrons bounce off the surface of the
specimen, revealing a three-dimensional
image. Deflected electrons are picked up by
a detector.
•  Best pictures - magnifications typically
10,000x, res. 10 nM.
•  Similar in idea to darkfield microscopy
Taxonomy
•  Taxonomy
–  The science of classifying organisms
–  Provides universal names for organisms
–  Provides a reference for identifying
organisms
Taxonomy
•  Systematics or phylogeny
–  The study of the evolutionary history of
organisms
•  All Species Inventory (2001-2025)
–  To identify all species of life on Earth Taxonomy
•  1735
Plant and Animal Kingdoms
•  1857
Bacteria & fungi put in the Plant Kingdom
•  1866
Kingdom Protista proposed for bacteria,
protozoa, algae, & fungi
•  1937
"Prokaryote" introduced for cells "without a nucleus"
•  1961
Prokaryote defined as cells in which nucleoplasm is not surrounded by a nuclear membrane
•  1959
Kingdom Fungi
•  1968
Kingdom Prokaryotae proposed
•  1978
Two types of prokaryotic cells found
The Three-Domain System
Table 10.1
The Three-Domain System
Figure 10.1
Scientific
Names
Source of Genus
Klebsiella pneumoniae
name
Honors Edwin Klebs
Source of Specific epithet
The disease
Pfiesteria piscicida
Honors Lois Pfiester
Disease in fish
Scientific binomial
Salmonella
Honors Daniel Salmon
typhimurium
Streptococcus pyogenes
Chains of cells
(strepto-)
Penicillium notatum
Tuftlike (penicill-)
Trypanosoma cruzi
Corkscrew-like
(trypano-, borer;
soma-body)
Stupor (typh-) in
mice (muri-)
Forms pus (pyo-)
Spores spread in
wind (nota)
Honors Oswaldo
Cruz
Taxonomic Hierarchy
Figure 10.5
Species Definition
•  Eukaryotic species:
–  A group of closely related organisms that breed
among themselves
•  Prokaryotic species:
–  A population of cells with similar characteristics
–  Clone: Population of cells derived from a single cell
–  Strain: Genetically different cells within a clone
•  Viral species: –  Population of viruses with similar characteristics that
occupies a particular ecological niche
Domain Eukarya
•  Animalia: Multicellular; no cell walls;
chemoheterotrophic
•  Plantae: Multicellular; cellulose cell walls;
usually photoautotrophic
•  Fungi: Chemoheterotrophic; unicellular or
multicellular; cell walls of chitin; develop from
spores or hyphal fragments
•  Protista: A catchall for eukaryotic organisms that
do not fit other kingdoms
Prokaryotes