Transcript Chapter 3

Chapter 3
Observing
Microorganisms
Through a
Microscope
© 2013 Pearson Education, Inc.
Copyright © 2013 Pearson Education, Inc.
Lectures prepared by Christine L. Case
Lectures prepared by Christine L. Case
Figure 3.2 Microscopes and Magnification.
Unaided eye
≥ 200 m
Light microscope
200 nm – 10 mm
Tick
Actual size
Scanning
electron
microscope
10 nm – 1 mm
Red blood cells
Transmission
electron
microscope
10 pm – 100  m
E. coli bacteria
T-even bacteriophages
(viruses)
Atomic force
microscope
0.1 nm – 10nm
DNA double helix
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Preparing Smears for Staining
 Live or unstained cells have little contrast with
the surrounding medium. Researchers do make
discoveries about cell behavior by observing live
specimens.
ANIMATION Microscopy and Staining: Overview
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Preparing Smears for Staining
 Staining: coloring the microbe with a dye that
emphasizes certain structures
 Smear: a thin film of a solution of microbes on
a slide
 A smear is usually fixed to attach the microbes to
the slide and to kill the microbes
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Simple Stains
 Simple stain: use of a single basic dye
 A mordant may be used to hold the stain or coat the
specimen to enlarge it
ANIMATION Staining
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Differential Stains
 Used to distinguish between bacteria
 Gram stain
 Acid-fast stain
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Gram Stain
 Classifies bacteria into gram-positive
or gram-negative
 Gram-positive bacteria tend to be killed by penicillin and
detergents
 Gram-negative bacteria are more resistant to antibiotics
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Gram Stain
Color of
Gram-Positive Cells
Color of
Gram-Negative 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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Figure 3.12 Gram staining.
Gram-positive
Gram-negative
Application of
crystal violet
(purple dye)
Application of
iodine (mordant)
Rod
(gram-negative)
Cocci
(gram-positive)
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Alcohol wash
(decolorization)
Application of
safranin (counterstain)
Acid-Fast Stain
 Stained waxy cell wall is not decolorized by
acid-alcohol
 Mycobacterium
 Nocardia
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Acid-Fast Stain
Color of
Acid-Fast
Color of
Non–Acid-Fast
Primary Stain:
Carbolfuchsin
Red
Red
Decolorizing Agent:
Acid-alcohol
Red
Colorless
Counterstain:
Methylene Blue
Red
Blue
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Figure 3.13 Acid-fast bacteria.
M. bovis
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Special Stains
 Used to distinguish parts of cells
 Capsule stain
 Endospore stain
 Flagella stain
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Negative Staining for Capsules
 Cells stained
 Capsule is not stained--negative stain
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Figure 3.14a Special staining.
Capsules
Negative staining
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Figure 3.14b Special staining.
Endospore
Endospore staining
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Figure 3.14c Special staining.
Flagellum
Flagella staining
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Table 3.3 A Summary of Various Stains and Their Uses
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