ch02 microscopy
TRANSCRIPT
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Copyright 2004 Pearson Education, Inc., publishing as Benjamin Cummings
Copyright 2004 Pearson Education, Inc., publishing as Benjamin Cummings
PowerPoint Lecture Slide Presentation prepared by Christine L. Case Modified by Nick Kapp
MicrobiologyB.E Pruitt & Jane J. Stein
AN INTRODUCTION9th EDITION
TORTORA FUNKE CASE
Chapter 3
Observing Microorganisms Through a
Microscope
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Copyright 2004 Pearson Education, Inc., publishing as Benjamin Cummings
Units of Measurement Table 3.1
1 m micrometer = 10-6 m = 10-3 mm 1 nm nanometer = 10-9 m = 10-6 mm
1000 nm = 1 m
0.001 m = 1 nm
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Microscopy: The Instruments
A simplemicroscope has onlyone lens.
Figure 1.2b
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Microscopy: The Instruments
In a compoundmicroscope the
image from theobjective lens ismagnified again bythe ocular lens.
Total magnification
=
objective lens v
ocular lens Figure 3.1b
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Microscopy: The Instruments
Resolution is the ability of the lenses todistinguish two points.
A microscope with a resolving power of 0.4 nm
can distinguish between two points 0.4 nm.
Shorter wavelengths of light provide greater
resolution
Resolving power=Wavelength of light used/2x
numerical aperture(a property of the lens).
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Microscopy: The Instruments
Refractive index isthe light-bending
ability of a medium.
The light may bendin air so much that it
misses the small
high-magnificationlens.
Immersion oil isused to keep light
from bending. Figure 3.3
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Brightfield Illumination
Dark objects arevisible against a
bright background.
Light reflected offthe specimen does
not enter theobjective lens.
Figure 3.4a, b
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Darkfield Illumination
Light objects are
visible against adark background.
Light reflected
off the specimenenters theobjective lens.
Figure 3.4a, b
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Phase-Contrast Microscopy
Accentuates diffractionof the light that passesthrough a specimen.
Direct and reflectedlight rays are combined
at the eye. Increasingcontrast
Figure 3.4c
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eren a n er erence on ras
Microscopy
Accentuatesdiffraction of
the light that
passes through
a specimen;uses twobeams of light.
Adding colorFigure 3.5
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Fluorescence Microscopy
Uses UV light.
Fluorescent
substances absorb
UV light and emitvisible light.
Cells may be stainedwith fluorescentdyes
(fluorochromes).Figure 3.6b
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Confocal Microscopy
Uses fluorochromes
and a laser light. The laser
illuminates each
plane in a specimento produce a 3-Dimage.
Figure 3.7
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Electron Microscopy
Uses electrons instead of light.
The shorter wavelength of electrons gives greaterresolution. Why?
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ransm ss on ec ron croscopy
(TEM) Ultrathin sections of specimens.
Light passes through specimen, then anelectromagnetic lens, to a screen or film.
Specimens may be stained with heavy metal salts.
Figure 3.8a
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ransm ss on ec ron croscopy
(TEM)
10,000-100,000v; resolution 2.5 nm
Figure 3.9
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Scanning Electron Microscopy (SEM)
An electron gun produces a beam of electrons thatscans the surface of a whole specimen.
Secondary electrons emitted from the specimenproduce the image.
Figure 3.9b
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Scanning Electron Microscopy (SEM)
1000-10,000v; resolution 20 nm
Figure 3.8b
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Scanning-Probe Microscopy
Scanning tunneling
microscopy uses ametal probe to scan
a specimen.
Resolution 1/100 ofan atom.
Figure 3.9a
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Scanning-Probe Microscopy
Atomic forcemicroscopy uses a
metal and diamond
probe inserted intothe specimen.
Produces 3-Dimages.
Figure 3.9b
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Preparation of Specimens forLight Microscopy
A thin film of a solution of microbes on aslide is a smear.
A smear is usually fixed to attach themicrobes to the slide and to kill themicrobes.
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Preparing Smears for Staining
Live orunstained cells
have little
contrast with thesurrounding
medium.However,researchers do
makediscoveries
about cell
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Preparing Smears for Staining
Stains consist of a positive and negative ion.
In a basic dye, the chromophore is a cation.
In an acidic dye, the chromophore is an anion.
Staining the background instead of the cell is
called negative staining.
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Simple Stains
Use of a single basic dye is called a simple stain.
A mordant may be used to hold the stain or coatthe specimen to enlarge it.
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Differential Stains: Gram Stain
The Gram stain classifies bacteria into gram-
positive and gram-negative. Gram-positivebacteria tend to be killed by
penicillin and detergents.
Gram-negativebacteria are more resistant toantibiotics.
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Differential Stains: Gram Stain
Color of
Gram + cells
Color of
Gram cells
Primary stain:
Crystal violet
Purple Purple
Mordant:
Iodine
Purple Purple
Decolorizing agent:
Alcohol-acetone
Purple Colorless
Counterstain:
Safranin
Purple Red
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Differential Stains: Gram Stain
Figure 3.11b
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Differential Stains: Acid-Fast Stain
Cells that retain a basic stain in the presence ofacid-alcohol are called acid-fast.
Nonacid-fast cells lose the basic stain when
rinsed with acid-alcohol, and are usually
counterstained (with a different color basic stain)to see them.
Figure 3.12
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Special Stains
Negative staining isuseful for capsules.
Heat is required todrive a stain intoendospores.
Flagella stainingrequires a mordant
to make the flagellawide enough to see.
Figure 3.13a-c
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Know the parts of the microscope
Know the types of light and electronic microscopes Power
What they are good for observing
What are stains used for?How do you do a gram stain