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Copyright © 2010 Pearson Education, Inc.Lectures prepared by Christine L. Case
Chapter 3
Observing Microorganisms
Through A Microscope
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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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Observing Microorganisms
Figure 3.2
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Learning Objectives
Chapter 3: Observing Microorganisms Through A Microscope
- List the metric units of measurement that are used for microorganisms.- Diagram the path of light through a compound microscope.- Define total magnification and resolution.- Identify a use for darkfield, phase-contrast, differential interference contrast, fluorescence,
confocal, two-photon, and scanning acoustic microscopy, and compare each with brightfield illumination.
- Explain how electron microscopy differs from light microscopy.- Identify one use for the TEM, SEM, and scanned-probe microscopes- Differentiate an acidic dye from a basic dye.- Explain the purpose of simple staining.- List the steps in preparing a Gram stain, and describe the appearance of gram-positive and
gram-negative cells after each step.- Compare and contrast the Gram stain and the acid-fast stain.- Explain why each of the following is used: capsule stain, endospore stain, flagella stain.
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Copyright © 2010 Pearson Education, Inc. Figure 3.2
Units of Measurement
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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Check Your Understanding If a microbe measures 10 μm in length, how long is
it in nanometers?
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Copyright © 2010 Pearson Education, Inc. Figure 1.2b
Microscopy: The Instruments
A simple microscope has only one lens
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Light Microscopy
Use of any kind of microscope that uses visible light to observe specimens
Types of light microscopy Compound light microscopy Darkfield microscopy Phase-contrast microscopy Differential interference contrast microscopy Fluorescence microscopy Confocal microscopy
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Copyright © 2010 Pearson Education, Inc. Figure 3.1a
The Compound Light Microscope
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Copyright © 2010 Pearson Education, Inc. Figure 3.1b
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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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 light-bending 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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Copyright © 2010 Pearson Education, Inc. Figure 3.3
Refraction in the Compound Microscope
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Copyright © 2010 Pearson Education, Inc. Figure 3.4a
ANIMATION Light Microscopy
Brightfield Illumination
Dark objects are visible against a bright background
Light reflected off the specimen does not enter the objective lens
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Copyright © 2010 Pearson Education, Inc. Figure 3.4b
Darkfield Illumination
Light objects are visible against a dark background
Light reflected off the specimen enters the objective lens
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Copyright © 2010 Pearson Education, Inc. Figure 3.4c
Phase-Contrast Microscopy
Accentuates diffraction of the light that passes through a specimen
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Copyright © 2010 Pearson Education, Inc. Figure 3.5
Differential Interference Contrast Microscopy Accentuates diffraction of the light that passes
through a specimen; uses two beams of light
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Copyright © 2010 Pearson Education, Inc. Figure 3.6b
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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Copyright © 2010 Pearson Education, Inc. Figure 3.7
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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Copyright © 2010 Pearson Education, Inc. Figure 3.8
Two-Photon Microscopy
Cells stained with fluorochrome dyes
Two photons of long- wavelength (red) light used to excite the dyes
Used to study cells attached to a surface Up to 1 mm deep
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Copyright © 2010 Pearson Education, Inc. Figure 3.9
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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Uses electrons instead of light The shorter wavelength of electrons gives greater
resolution
ANIMATION Electron Microscopy
Electron Microscopy
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Copyright © 2010 Pearson Education, Inc. Figure 3.10a
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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Copyright © 2010 Pearson Education, Inc. Figure 3.10a
Transmission Electron Microscopy (TEM)
10,000–100,000; resolution 2.5 nm
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Copyright © 2010 Pearson Education, Inc. Figure 3.10b
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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Copyright © 2010 Pearson Education, Inc. Figure 3.10b
Scanning Electron Microscopy (SEM)
1,000–10,000; resolution 20 nm
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Copyright © 2010 Pearson Education, Inc. Figure 3.11a
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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Copyright © 2010 Pearson Education, Inc. Figure 3.11b
Scanned-Probe Microscopy
Atomic force microscopy (AFM) uses a metal- and-diamond probe inserted into the specimen.
Produces three-dimensional images.
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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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Live or unstained cells have little contrast with the surrounding medium. Researchers do make discoveries about cell behavior by observing live specimens.
Figures B and C
ANIMATION Microscopy and Staining: Overview
Preparing Smears for Staining
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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 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
Simple Stains
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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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Color of Gram-positive cells
Color ofGram-negative cells
Primary stain:Crystal violet
Purple Purple
Mordant:Iodine
Purple Purple
Decolorizing agent:Alcohol-acetone
Purple Colorless
Counterstain:Safranin
Purple Red
Gram Stain
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Copyright © 2010 Pearson Education, Inc. Figure 3.12b
Micrograph of Gram-Stained Bacteria
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Acid-Fast Stain
Stained waxy cell wall is not decolorized by acid-alcohol
Mycobacterium Nocardia
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Color of Acid-fast
Color ofNon–Acid-fast
Primary stain:Carbolfuchsin
Red Red
Decolorizing agent:Acid-alcohol
Red Colorless
Counterstain:Methylene blue
Red Blue
Acid-Fast Stain
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Copyright © 2010 Pearson Education, Inc. Figure 3.13
Acid-Fast Bacteria
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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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Special Stains
Used to distinguish parts of cells Capsule stain Endospore stain Flagella stain
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Copyright © 2010 Pearson Education, Inc. Figure 3.14a
Negative Staining for Capsules
Cells stained Negative stain
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Copyright © 2010 Pearson Education, Inc. Figure 3.14b
Endospore Staining
Primary stain: Malachite green, usually with heat Decolorize cells: Water Counterstain: Safranin
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Copyright © 2010 Pearson Education, Inc. Figure 3.14c
Flagella Staining
Mordant on flagella Carbolfuchsin simple stain