Microscopy

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Microscopy

• Kathy Huschle
• Northland Community and Technical College

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Microscopy

• microscope
• produces enlarged images
• studies the morphology (what it looks like) of a
  microorganism

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Microscopy

• microscopy includes
• magnification
• resolution
• contrast

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Magnification

• magnification
• a result of refraction or bending of light rays

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Resolution

• resolution
• ability to distinguish detail
• will allow you to observe one layer at a time

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Contrast

• contrast
• visible shades in a specimen
• contrast is often enhanced by staining

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Light Microscopes

• Bright-field Microscope
• field of view is brightly illuminated with
  visible light
• also known as compound microscope

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Fluorescence Microscope

• Fluorescence Microscope
• use with fluorescent stains
• emit light at a different wavelength
• different wavelengths have different colors

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Fluorescence Microscope

• dyes can be chemically linked specific chemical
  targets
• target can be visualized and quickly and
  positively identified
• allows for rapid diagnosis

Click icon to use a virtual fluorescence
scope. Click Try the Simulator
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Fluorescence Microscope
Image of cell division taken by fluorescence
microscope
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Dark Field Microscope

• Dark-field Microscope
• enhance contrast without staining
• most staining will kill the microorganism
• allows for the viewing of live specimens
• special condenseris used
• focuses light at an angle
• reflected off specimen

Treponema pallidum image from Dark Field
Microscope
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Phase Contrast Microscope

• Phase Contrast Microscope
• contrast without staining
• difference in density produces difference in
  light
• brings direct and reflected light rays together
  to form an image of the specimen

image of cell division taken by phase contrast
microscope
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Phase Contrast Microscope

• allows for observation of living organisms

Click icon to use a virtual phase contrast
scope. Click Try the Simulator
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Interference Microscope

• Interference Microscope
• two light waves combined
• beam is split
• one part through the specimen
• one part around
• recombined

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Interference Microscope

• specimen looks three dimensional

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Confocal Scanning Laser Microscope

• constructs a 3-dimensional image
• specimen is stained with fluorescent dye

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Confocal Scanning Laser Microscope

• specimen is illuminated one plane at a time
• computer processes the images into 2 and 3
  dimensional images

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Electron Microscopy

• electron beam is used in the place of a beam of
  light
• electromagnets control focus, illumination and
  magnification
• higher magnification up to 100,000X

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Electron Microscopy

• electron beam must be contained in a vacuum to
  eliminate disturbance from air molecules
• large, bulky unit
• specimen must be killed
• special preparation process
• problem with artifacts

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Transmission Electron Microscope

• TEM
• used for observing layers of cells

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Transmission Electron Microscope

• 10,000 100,000 X magnification

rubber particle magnified 100,000 times
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Transmission Electron Microscope
Click icon to use a virtual Transmission
electron scope. Click Try the Simulator
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Scanning Electron Microscope

• SEM
• used for surface features, not internal features
• knocks electrons out of specimen surface
• collected for image

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Scanning Electron Microscope

• SEM
• produces a 3-D image
• 1,000 10,000 X magnification

image of table salt taken with an SEM
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Scanning Electron Microscope
Click icon to use a virtual Scanning electron
scope. Click Try the Simulator
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Contrast Staining

• stain is a salt with a negative and positive
  charged ion
• color portion is a positive charge and will be
  attracted to the negative charged microbial cell

Enterobacter
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Contrast Staining

• stain
• increases contrast between specimen and
  background
• most microorganisms are colorless and difficult
  to see

Unstained Halophile
Stained Halophile
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Contrast Staining

• simple stains
• positive staining
• cells are dark against light background
• makes cell shapes and arrangements visible

yeast stained with methylene blue stain
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Contrast Staining

• negative staining
• clear microorganisms against a dark background
• better view of microbial shape due to lack of
  distortion
• stain is not taken up by the microorganism
• very useful with microorganisms with capsules

negatively stained mixed bacteria culture
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Differential Staining

• differential staining
• multiple stains
• differentiate bacteria according to their
  reaction to the staining process
• differentiation is based on differences in cell
  structure
• Gram stain
• acid fast stain

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Differential Staining

• Gram Stain
• most widely used procedure
• process uses a stain, wash, counterstain

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Differential Staining

• Gram Stain
• Gram positive bacteria retain the purple stain
• Gram negative bacteria appear pink after the
  counterstain

Gram - Gram
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Differential Staining

• acid-fast staining
• waxy chemical in the cell wall allows the
  bacteria to retain the stain

Cryptosporidum muris
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Differential Staining

• acid-fast staining
• makes Mycobacterium ( causative agent for
  tuberculosis) easy to recognize in sputum, amidst
  numerous organisms

sputum sample containing M. tuberculosis
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Special Stains

• stains that color only certain parts of bacteria
• endospore staining
• reveals the presence of endospores
• produced by few bacteria
• Clostridium and Bacillus

A is the microorganism, B indicates the endospore
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Special Stains

• flagella staining
• adheres to an enlarges the flagella for ease of
  observation

flagella
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Bacteria MorphologyShape

• 3 basic shapes
• coccus
• bacilus
• spiral

spiral
coccus
bacillus
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Bacteria Morphologyshape

• coccus
• primarily spherical in shape
• can be oval, elongated or flattened on one side
• plural cocci

Enterococcus sp.
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Bacteria Morphologyshape

• bacillus
• rod shaped
• plural bacilli

Bacillus anthracis
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Bacteria Morphologyshape

• spiral
• one or more twists
• never straight

Treponema pallidum
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Bacterial MorphologyArrangement

• single

yeast cells
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Bacterial Morphologyarrangement

• diplococci or diplobacilli

Neisseria gonorrhoeae
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Bacterial Morphologyarrangement

• chains of coccus or bacillus

Streptococcus sp.
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Bacterial Morphologyarrangement

• clusters