BIOL 3702 Lecture Outline

Chapter 2 - The Study of Microbial Structure: Microscopy and Specimen Preparation

The Light Microscope

The size of most microbes are typically described in terms of micrometers (m) Compound microscopes are commonly used to clearly visualize microbes including the
following types:
Bright field Dark field Phase contrast Differential interference contrast Fluorescent

Bright field microscopy

Simple, relatively inexpensive Can observe living microbes if they have enough contrast, but typically specimens

consisted of dead, stained cells image on a light background

Uses a series of lenses termed objectives Images illuminated by a light source Most important property of a microscope is resolution
The The The

ability of a lens to separate or distinguish between small objects that are close together smaller the wavelength of light, the greater the resolution light microscope has a maximum theoretical resolution of 0.2 m

Magnification increases the size of an object, but is limited by resolution At 1000X magnification, need to replace the air between the coverslip/slide and the

objective with immersion oil

Increases Results

amount of normally refracted light into objective

in increased resolution

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BIOL 3702 Lecture Outline

Chapter 2

Dark-field microscopy
Can observe living, unstained cells Uses a hollow cone of light focused in such a way that only the light reflected off the

edges of a specimen is visible

The area surrounding the specimen appears dark, hence, dark field Can view internal structures of eucaryotes

Phase-contrast microscopy
Can observe living, unstained cells Uses phase shifted light Can detect internal details in both procaryotes and eucaryotes

Differential interference contrast microscopy

Detects differences in refractive indices and thickness of image Uses polarized light beams passed through a specimen at right angles Can use live, unstained specimen and view a three-dimensional image

Fluorescent Microscopy
Detects light emitted from a specimen that is typically stained with a special dye

molecule termed a fluorochrome

Fluorochrome releases energy in the form of light when excited by fluorescent light Usually uses a mercury vapor lamp or similar light source Images appear colored; color depends upon the fluorochrome used Color of image depends upon the fluorochrome used Used frequently as a diagnostic tool

Specimen Preparation

Microbes must often be fixed and stained to:

Increase visibility Accentuate morphological features Preserve them for future study

Fixation
Process by which internal and external structures of cells and microbes are preserved

and fixed into position

Two types of fixation:
Heat

fixation fixation
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Chemical

BIOL 3702 Lecture Outline

Heat fixation preserves overall morphology, but not internal structures

Chapter 2

Chemical fixation protects the fine cellular substructures and the morphology of larger,

more delicate microbes

Simple stain
Use

of a single basic dye to stain a microbe to determine size, shape, and arrangement of the microbes being observed

Used

Differential staining
Divide bacteria into different groups based on their staining patterns Gram stain - developed by Christian Gram
Most

employed staining method in bacteriology bacteria into Gram-positive and Gram-negative groups stain - crystal violet with ethanol or acetone with safranin bacteria - purple/blue bacteria - red/pink steps mordant Grams iodine

Divides Four

Basic Add

Decolorize

Counterstain

Results
Gram-positive

Gram-negative Gram Gram

variable non-reactive

Acid fast stain (Ziehl-Neelsen)

Some

bacterial species, particularly Mycobacteria, do not bind simple stains specimen with a mixture of basic fuchsin and phenol with acid alcohol (mycobacteria do not easily decolorize) with methylene blue

Procedure
Heat

Decolorize

Counterstain

Results
Acid-fast

microbes stain red microbes stain blue

Non-acid-fast

Technique

is important presumptive diagnostic tool for tuberculosis and leprosy

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BIOL 3702 Lecture Outline

Chapter 2

Staining specific structures

Negative staining
Reveals

the presence of capsules by the exclusion of the dye

Procedure
Add

India ink or nigrosin dye to air-dried smear of bacteria counterstain to visualize the cell are highly resistant to staining, but resist decolorization once stained

Optional

Endospore stain (Schaeffer-Fulton)

Endospores Procedure
Heat

with malachite green and counterstain with safranin stain blue/green

Wash

Results
Endospores Vegetative

cell stains red/purple

Flagella stain - made visible by increasing thickness

Electron Microscopy

Resolution of images are far greater than those obtained by light microscopy Electrons are used instead of light to illuminate the specimen Two general types of electron microscopy
Transmission (TEM) Scanning (SEM)

Specimen preparation for TEM and SEM is tedious and labor intensive as well as markedly
different than that employed in light microscopy

Transmission electron microscopy

Electrons, which behave in a physical manner similar to light, have a wavelength about

100,000 times shorter

Resolution is more than 1000 times greater (down to 5) with magnifications typically

near 100,000X

Transmission electron microscopy

Electrons gun generates an electron beam within a vacuum Beam is controlled by electromagnets which direct the electrons to the specimen Electrons pass through or are scattered by the specimen form an image on a fluorescent

screen or photographic film

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BIOL 3702 Lecture Outline

Resulting image:
Scattered Electrons

Chapter 2

electrons form dark areas that pass through form light areas

Scanning electron microscopy

Electrons beam, generated within a vacuum, scans a specimen and is controlled by

electromagnets
Electrons cause the release of secondary electrons that are trapped by a detector Signals are amplified to form a three-dimensional image of surface features

Specimen preparation for electron microscopy

TEM
Thin

sections are then usually stained with a heavy metal salt (e.g., lead, uranium, are mounted onto tiny copper grids before being viewed within the TEM

etc.)
Sections Special

viewing methods

Negative staining - apply a heavy metal acid that does not penetrate the specimen, creates a dark background useful to study the structure of small objects like viruses Shadowing - coating a specimen with a thin film of metal at an angle thereby causing shadows Freeze etch - fracturing a frozen cell along planes of weakness (as in cutting a diamond), then shadowing the frozen surface with a film of heavy metal to reveal three-dimensional images of internal structures

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