Transcript Chapter 3 PowerPoint

Chapter 3
Observing
Microorganisms
Through A
Microscope
Copyright © 2010 Pearson Education, Inc.
Lectures prepared by Christine L. Case
Q&A
 Acid-fast staining of a
patient’s sputum is a
rapid, reliable, and
inexpensive method to
diagnose tuberculosis.
What color would
bacterial cells appear if
the patient has
tuberculosis?

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Look for the answer in the chapter
.
Observing Microorganisms
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Figure 3.2
Units of Measurement
Learning Objectives
3-1 List the metric units of measurement that are
used for microorganisms.
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Units of Measurement
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1 µm = 10–6 m = 10–3 mm
1 nm = 10–9 m = 10–6 mm
1000 nm = 1 µm
0.001 µm = 1 nm
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Figure 3.2
Check Your Understanding
 If a microbe measures 10 μm in length, how long is
it in nanometers? 3-1
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Microscopy: The Instruments
Learning Objectives
3-2 Diagram the path of light through a compound
microscope.
3-3 Define total magnification and resolution.
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Microscopy: The Instruments
 A simple microscope has only one lens
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Figure 1.2b
Light Microscopy
 Use of any kind of microscope that uses visible light
to observe specimens
 Types of light microscopy
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Compound light microscopy
Darkfield microscopy
Phase-contrast microscopy
Differential interference contrast microscopy
Fluorescence microscopy
Confocal microscopy
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The Compound Light Microscope
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Figure 3.1a
Compound Light Microscopy
 In a compound
microscope, the image
from the objective lens
is magnified again by
the ocular lens
 Total magnification =
objective lens  ocular
lens
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Figure 3.1b
Compound Light Microscopy
 Resolution is the ability of the lenses to distinguish
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
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Compound Light Microscopy
 The refractive index is a measure of the lightbending ability of a medium
 The light may bend in air so much that it misses the
small high-magnification lens
 Immersion oil is used to keep light from bending
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Refraction in the Compound Microscope
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Figure 3.3
Check Your Understanding
 Through what lenses does light pass in a compound
microscope? 3-2
 What does it mean when a microscope has a
resolution of 0.2 nm? 3-3
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Microscopy: The Instruments
Learning Objectives
3-4 Identify a use for darkfield, phase-contrast,
differential interference contrast, fluorescence,
confocal, two-photon, and scanning acoustic
microscopy, and compare each with brightfield
illumination.
3-5 Explain how electron microscopy differs from light
microscopy.
3-6 Identify one use for the TEM, SEM, and scannedprobe microscopes.
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Brightfield Illumination
 Dark objects are visible
against a bright background
 Light reflected off the
specimen does not enter
the objective lens
ANIMATION Light Microscopy
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Figure 3.4a
Darkfield Illumination
 Light objects are visible
against a dark
background
 Light reflected off the
specimen enters the
objective lens
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Figure 3.4b
Phase-Contrast Microscopy
 Accentuates diffraction
of the light that passes
through a specimen
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Figure 3.4c
Differential Interference Contrast
Microscopy
 Accentuates diffraction of the light that passes
through a specimen; uses two beams of light
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Figure 3.5
Fluorescence Microscopy
 Uses UV light
 Fluorescent
substances absorb UV
light and emit visible
light
 Cells may be stained
with fluorescent dyes
(fluorochromes)
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Figure 3.6b
Confocal Microscopy
 Cells stained with
fluorochrome dyes
 Short wavelength
(blue) light used to
excite the dyes
 The light illuminates
each plane in a
specimen to produce
a three-dimensional
image
 Up to 100 µm deep
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Figure 3.7
Two-Photon Microscopy
 Cells stained with
fluorochrome dyes
 Two photons of longwavelength (red) light
used to excite the
dyes
 Used to study cells
attached to a surface
 Up to 1 mm deep
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Figure 3.8
Scanning Acoustic Microscopy (SAM)
 Measures sound
waves that are
reflected back from
an object
 Used to study cells
attached to a
surface
 Resolution 1 µm
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Figure 3.9
Electron Microscopy
 Uses electrons instead of light
 The shorter wavelength of electrons gives greater
resolution
ANIMATION Electron Microscopy
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Transmission Electron Microscopy (TEM)
 Ultrathin sections of
specimens
 Light passes through
specimen, then an
electromagnetic lens,
to a screen or film
 Specimens may be
stained with heavy
metal salts
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Figure 3.10a
Transmission Electron Microscopy (TEM)
 10,000–100,000; resolution 2.5 nm
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Figure 3.10a
Scanning Electron Microscopy (SEM)
 An electron gun
produces a beam of
electrons that scans
the surface of a
whole specimen
 Secondary electrons
emitted from the
specimen produce
the image
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Figure 3.10b
Scanning Electron Microscopy (SEM)
 1,000–10,000; resolution 20 nm
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Figure 3.10b
Scanned-Probe Microscopy
 Scanning tunneling microscopy (STM) uses a
metal probe to scan a specimen
 Resolution 1/100 of an atom
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Figure 3.11a
Scanned-Probe Microscopy
 Atomic force microscopy (AFM) uses a metaland-diamond probe inserted into the specimen.
 Produces three-dimensional images.
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Figure 3.11b
Check Your Understanding
 How are brightfield, darkfield, phase-contrast, and
fluorescence microscopy similar? 3-4
 Why do electron microscopes have greater resolution than
light microscopes? 3-5
 For what is TEM used? SEM? Scanned-probe microscopy?
3-6
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Preparation of Specimens for Light
Microscopy
Learning Objectives
3-7 Differentiate an acidic dye from a basic dye.
3-8 Explain the purpose of simple staining.
3-9 List the steps in preparing a Gram stain, and
describe the appearance of gram-positive and
gram-negative cells after each step.
3-10 Compare and contrast the Gram stain and the
acid-fast stain.
3-11 Explain why each of the following is used:
capsule stain, endospore stain, flagella stain.
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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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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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Figures B and C
Preparing Smears for Staining
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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
 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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Micrograph of Gram-Stained Bacteria
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Figure 3.12b
Check Your Understanding
 Why doesn’t a negative stain color a cell? 3-7
 Why is fixing necessary for most staining
procedures? 3-8
 Why is the Gram stain so useful? 3-9
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Acid-Fast Stain
 Stained waxy cell wall is not decolorized by acidalcohol
 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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Acid-Fast Bacteria
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Figure 3.13
Q&A
 Acid-fast staining of a
patient’s sputum is a
rapid, reliable, and
inexpensive method to
diagnose tuberculosis.
What color would
bacterial cells appear if
the patient has
tuberculosis?
Copyright © 2010 Pearson Education, Inc.
Special Stains
 Used to distinguish parts of cells
 Capsule stain
 Endospore stain
 Flagella stain
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Negative Staining for Capsules
 Cells stained
 Negative stain
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Figure 3.14a
Endospore Staining
 Primary stain: Malachite green, usually with heat
 Decolorize cells: Water
 Counterstain: Safranin
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Figure 3.14b
Flagella Staining
 Mordant on flagella
 Carbolfuchsin simple stain
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Figure 3.14c
Check Your Understanding
 Which stain would be used to identify microbes in the genera
Mycobacterium and Nocardia?
3-10
 How do unstained endospores appear?
Stained endospores? 3-11
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