Transcript A TOUR OF THE CELL

Microscopy
Observing Microorganisms Through a
Microscope
Units of Measurement
Microorganisms are so small that metric
prefixes may be unfamiliar
centi = 1/100
milli = 1/1000
micro = 1/1,000,000
nano = 1/1,000,000,000
or
or
or
or
10-1
10-2
10-6
10-9
The Instruments
Compound Light Microscope
Electron Microscopes
Darkfield Microscopy
Scanning Electron Microscopy
Phase Contrast Microscopy
Transmission Electron Microscopy
Differential Interference Contrast Microscopy
Atomic Force Electron Microscopy
Fluorescence Microscopy
Confocal Microscopy
Compound Light Microscope
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Uses visible light
Has at least 2 sets of lenses
Can achieve maximum 2000X
magnification
Resolution of objects as small as
0.2 m
Light Microscopy
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In a light microscope visible light
passes through the specimen and then
through glass lenses.
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The lenses refract light such that the
image is magnified into the eye or a video
screen.
Light Microscopes
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Microscopes vary in magnification and
resolving power.
Magnification is the ratio of an object’s image
to its real size.
Resolving power is a measure of image clarity.
 It is the minimum distance two points can be
separated and still viewed as two separate
points.
 Resolution is limited by the shortest
wavelength of the source, in this case light.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Resolution of Light Microscopes
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The minimum resolution of
a light microscope is about
2 microns, the size of a
small bacterium
Light microscopes can
magnify effectively to
about 1,000 times the size
of the actual specimen.
 At higher magnifications,
the image blurs. Fig. 7.1
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Brightfield Illumination
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Usual operations
Specimens must be stained
for viewing
Best magnification and
resolution with the oil
immersion objective
Oil has same refractive index
as glass
Darkfield Microscopy
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Only light reflected from specimen enters objective lens
Organism appears light against a dark field
Useful for examining
Live organisms
 Microorganisms which cannot be stained by standard methods
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Treponema pallidum, the causative agent of syphilis
Phase-Contrast and Differential Interference
(DIC) Microscopy
Phase-Contrast
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Uses wave nature of light
One set of light rays are direct and
one set are reflected
Makes detailed images of internal
structure of living microorganisms
possible
Image in greyscale
DIC
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Uses differences in refractive
indices
Uses 2 beams of light
Resolution higher
Brightly colored image
Image appears nearly threedimensional
Electron micrographs
Fluorescence Microscopy
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When illuminated with short  light
some dyes emit light with longer 
Enables viewing of cells located on
an opaque surface such as a soil
particle
When illuminated with UV or
halogen light source preparations
glow
Bovine pulmonary artery endothelial cells.
Photometrics, Ltd.
Fluorescent stain of cell
Confocal Microscopy
3-D confocal microscopy of Salmonella-infected macrophage (green) with XY-slice showing bacteria (red) inside the cell
•Preparations also stained with fluorochromes
•Exceptionally clear two-dimensional images
•Three-dimensional images obtained by computer construct
Electron Microscopy
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Beam of electrons has shorter  so gives better resolution
than visible light
Electromagnetic lenses rather than glass
Done in a vacuum
Can resolve to 0.5nm and magnify up to 100,000 times.
Specimen must be dry….dead
Transmission Electron Microscopy
(TEM)
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Resolves objects as close as
2.5nm
Magnification 10,000 to
100,000X
Ultra-thin sections
Specimens must be
dehydrated
Preparation of specimen may
generate artifacts
Lambda Bacteriophage DNA
(TEM x153,000)
Scanning Electron Microscopy
(SEM)
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Resolves objects as close as
20 nm
Magnification between 1,000
and 10,000X
Whole specimens
3-dimensional view of
specimen
Specimen dehydrated
Slime Mold Fruiting Structure, Lamproderma sp.
(SEM x290)
Scanning Tunneling Atomic Force
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Thin metal probe scans specimen
Resolving power much greater
than electron microscopes
no special specimen preparation
detailed views of silicon chips &
DNA molecule
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Metal and diamond probe forced
down along surface of specimen
3-dimensional image
no special preparation of specimen
is required
views of detailed structure of
biological molecules
Preparation of Microscopy Specimens
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Microorganisms must be spread over the surface of a slide
(smear)
Microorganisms must be attached to the slide (fixed)
Microorganisms must be colored (stained)
Making the Smear and Fixing It
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Put a small amount of organism
into a drop of water on a clean
microscope slide & spread .
When dry pass through the
flame of a Bunsen burner or
flood with methyl alcohol
Stains
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Dyes
Negative Stain
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Simple Stains
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stains the background not the organism
everything stained a single color
Differential Stains
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distinguish among bacteria based on particular characteristics
Characteristics of Dyes
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Basic Dyes
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Acidic Dyes
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Chromophore is in the positive
ion
Used to stain most bacteria
Used alone as simple stains in
combination for differential
stains
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Chromophore is in the negative
ion
Used in negative staining & for
staining nuclear material
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Gram Stain
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The Gram Stain is the single most important test in microbiology. The
principal utility of the Gram Stain rests on its speed and simplicity.
Most bacteria may be divided in two groups by this procedure
developed by the Danish physician Hans Christian Gram to
differentiate pneumococci from Klebsiella pneumonia
difference between Gram-positive and Gram-negative bacteria is in the
structure of the cell wall
Procedure
Results
G+ cocci
G- rods
Websites with more samples of gram stained bacteria
GRAM STAINED IMAGES OF MEDICALLY IMPORTANT BACTERIA
Loyola University Medical Center
http://www.meddean.luc.edu/lumen/DeptWebs/microbio/med/gram/slides.htm
GRAM STAIN TUTORIAL
http://www.courses.ahc.umn.edu/pharmacy/5825/GSPage05.html
Acid Fast Stain
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Acid Fast Staining is used primarily for detection of organisms with a thick outer coat
composed of true waxes, mycolic acids and phosphatides
Mycobacteria are not decolorized and retain the stain, appearing pink under the light
microscope (hence are 'fast', like color-fast clothes).
Acid Fast Stain can also be used to identify several protozoa, such as Cryptosporidium and
Isospora belli. These two coccidia have recently acquired greater clinical significance because
of their widespread occurrence in immuno- compromised patients, such as those infected
with HIV.
Special Stains
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Spore Stain
Bacillus subtilis
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Capsule Stain
Streptococcus pneumoniae
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Flagella Stain
Pseudomonas aeruginosa