MICROSCOPY AND STAINING

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MICROSCOPY AND STAINING

• CHAPTER 3

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Metric Units
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Light Properties

• Wavelength

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polarity
Light is a wave
Filters can block waves in off axis planes
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Waves can be added




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Light Properties

• Resolution

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Wavelength/Resolution Interaction
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Light Properties

• Reflection
• Transmission

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Light Properties

• Absorption
• Refraction

bending
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Light Microscopy Types

• Compound
• Bright Field

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Oil immersion
Oil with intermediate refractive index
without
With oil
Some info lost
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Microscopy Dark Field
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Microscopy Phase Contrast

• Dual Beam

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Phase contrast

• Phase-contrast microscopy was invented in 1936
  by Frits Zernike, a Dutch mathematical physicist.
  It is based on the principle that cells differ in
  refractive index (a factor by which light is
  slowed as it passes through a material) from
  their surroundings. Light passing through a cell
  thus differs in phase from light passing through
  its surroundings. This subtle difference is
  amplified by a device in the objective lens of
  the phase-contrast microscope called the phase
  ring, resulting in a dark image on a light
  background (Figure 2.5b). The ring consists of a
  phase platethe key discovery of Zernikethat
  amplifies the minute variation in phase.
  Zernikes discovery of differences in contrast
  between cells and their background stimulated
  other innovations in microscopy, such as
  fluorescence and confocal microscopy (discussed
  below). For his invention of phase-contrast
  microscopy, Zernike was awarded the 1953 Nobel
  Prize in Physics.

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Microscopy DIC

• Differential Interference Contrast

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DIC differential interference contrast
Similar to phase contrast, but input light is
polarized
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Microscopy Fluorescence

• Ultraviolet light

flourescein
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Advantages of fluorescence
Can use specialized chemical probes that target
specific features and then tag with fluorescent
dyes
Downside must use expensive filters and excitory
frequencies
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Microscopy Confocal

• Confocal

Allows 3 dimensional viewing
Allows multiple dyes to be overlaid
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Confocal microscopy
Allows 3 dimensions
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Combined confocal and fluorescence
Antibody labeling
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Microscopy Imaging

• Digital

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Fig. 2-15
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Electron Microscopy

• Transmission (TEM)
• Scanning (SEM)
• Scanning Tunneling (STM)

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TEM
Most popular for bacteria. Allows imaging
internal features, but requires heavy
metal staining.
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Electron Microscopy Images
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Microscopy Techniques

• Wet Mounts
• Smears
• Staining

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Fig. 2-3
Spread culture in thin film over slide
Dry in air
I. Preparing a smear
Flood slide with stain rinse and dry
Pass slide through flame to heat fix
II. Heat fixing and staining
100?
Slide
Oil
Place drop of oil on slide examine with
100? objective lens
III. Microscopy
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Staining cells - increases contrast
Simple stain - one dye - shows size, shape,
and arrangement
Methylene blue -
yeast
Cheek cell
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Common stains
Safranin (basic, charge)
red
Crystal violet
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Differential stains
Use multiple dyes or dyes that interact with
organisms differently.
Primary stain / counterstain
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Gram Stain

• Gram Stain The single most important stain in
  microbiology. Set the initial taxonomy of
  bacteria.
• Crystal violet (basic stain)

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Gram Stain
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Acid Fast Stain
Carbol-fuchsin stains acid fast organisms
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Acid Fast

• The Ziehl-Neelsen stain, also known as the
  acid-fast stain, was first described by two
  German doctors Franz Ziehl (1859 to 1926), a
  bacteriologist and Friedrich Neelsen (1854 to
  1894), a pathologist. It is a special
  bacteriological stain used to identify acid-fast
  organisms, mainly Mycobacteria. Mycobacterium
  tuberculosis is the most important of this group,
  as it is responsible for the disease called
  tuberculosis (TB). It is helpful in diagnosing
  Mycobacterium tuberculosis since its lipid rich
  cell wall makes it resistant to Gram stain. It
  can also be used to stain few other bacteria like
  Nocardia. The reagents used are Ziehl-Neelsen
  carbolfuchsin, acid alcohol and methylene blue.

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Acid Fast of Mycobacterium tuberculosis
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Negative Stain
India ink or nigrosin
Sometimes referred to as capsular stain
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Flagellar Stain
Salmonella typhimurium
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Endospore Stain

• Used on spore forming bacteria such as Bacillus
  sp.

Malachite green stains spores