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7/27/2019 Observing Microorganisms Through a Microscope
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Unit of Measure
Bacteria are VERY small, thats why this
is micro biology
The standard unit of measure in
microbiology is the MICROMETER (m)
A micrometer is 10-6 or .000001M
To see something this small you need touse a microscope and also color (stain)
the cells to see them
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Microscope
Because bacteria are so small a
microscope is the essential tool in
microbiology
Light microscope uses visible light to
observe bacteria
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Total Magnification
Ocular
(Eye Piece)
Objective Total
Magnification
10X 4X 40X
10X 10X 100X
10X 40X 400X
10X 100X (oil) 1000X
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Resolution
Ability of the lens to distinguish fine detail
How close together can you distinguishtwo points as separate?
Because bacteria are so small goodresolution is important
Resolution is DIRECTLY related to light in
the following way: The SHORTER thewavelength of light the GREATER theresolution
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More Resolution
To get the best resolution, use the
SHORTEST wavelength of visible light
that you can
Our microscopes use blue wavelength
light to maximize resolution
Using blue light we can get a resolution of
about .9 micrometers (m)
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Visible Spectrum
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Spectrum
Average wavelength of visible light is
.55m
Red light wavelength is .68m, violet light
is .42m, blue light is .48m
Which light is best to use? The one with
shortest wavelength
Using a shorter wavelength of light in the
blue range give better resolution
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Size Matters
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Why Use Oil?
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Smears and Staining
Bacteria must be stained (dyed) so theycan be seen with the microscope
Before staining a smear must be made
A smear is just a film of bacteria on aglass slide
After the smear dries it is heat fixed, this
Kills the bacteria Helps adhere the cells to the slide
Makes the cells more receptive to the dye
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Stains
Stains are dyes
Stains carry either a positive charge (basic dyes)
or a negative charge (acidic dyes)
Bacteria typically carry a slight negative chargeon the cell surface so they attract a basic dye
Most of the stains used in the lab are basic dyes
A negative stain uses acidic dyes that do notstain the cell but rather the background
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Staining
Positive
staining: thedye sticks to thespecimen togive it color
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Staining Techniques
Simple Stain
Uses only one basic dye
Provides basic
information about cellshape and arrangement
Differential Stain
Uses more than one dye
These procedures react
differently with differentkinds of bacteria
Helps distinguish
between different kinds of
bacteria
Most common and
important differential stain
is the GRAM STAIN
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Simple Stains
Require only a
single dye
Examples include
malachite green,crystal violet, basic
fuchsin, and
safranin
All cells appear the
same color but can
reveal shape, size,
and arrangement
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Differential Stains
Use two differentlycolored dyes, the
primary dye and the
counterstain
Distinguishesbetween cell
types or parts
Examples
include Gram,
acid-fast, and
endospore stains
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Gram Staining
The most universal
diagnostic staining
technique for bacteria
Differentiation ofmicrobes as gram
positive (purple) or
gram negative (red)
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Gram Stain
Most important differential staining
technique
Differentiates all bacteria based on cell
wall composition
Bacteria are either Gram + and stain blue
or Gram- and stain red
Gram stain is usually the first step in
identifying an unknown bacteria
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Gram Stain
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Gram stain
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Endospore Stain
Dye is forced by heat
into resistant bodies
called spores or
endospores Distinguishes
between the stores
and the cells they
come from (thevegetative cells)
Significant in medical
microbiology
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Special Stains
Used to
emphasize
certain cell parts
that arentrevealed by
conventional
staining methods
Examples:capsule staining,
flagellar staining
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