MICROSCOPY AND STAINING

Leeuwenhoek’s Investigation

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Metric Units

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Light Properties

Wavelength

http://eyetricks.com/0101.htm

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Light Properties

Reflection

Transmission

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Light Microscopy Types

CompoundBright Field

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Light Properties

Resolution

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Wavelength/Resolution Interaction

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Light Properties

Absorption

Refraction

http://interactagram.com/physics/optics/refraction/

Effect of Immersion Oil

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Disappearing Diamond

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Diffraction

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Diffraction

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Microscopy — Dark Field

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Microscopy — Phase Contrast

Dual Beam

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Microscopy — DIC (Nomarsky)

Differential Interference Contrast

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Microscopy — Fluorescence

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Microscopy — Confocal

Confocal

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Microscopy Imaging

Digital

Same Image – Different Techniques

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Electron Microscopy Transmission

(TEM or EM) Scanning (SEM)

Scanning Tunneling (STM)

Atomic Force (AFM)

Scanning Acoustic (SAM)

http://micro.magnet.fsu.edu/primer/virtual/virtual.html*animation

Freeze - fracture

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SEM Shadow Casting

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SEM freeze-etch

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Electron Microscopy Images

Electron Microscopy Images

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STM

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AFM

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Microscopy Techniques Wet Mounts Smears Drop

Simple Stain

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• Provide basic contrast for light compound microscope• Anionic = acidic dyes:

• eosin• picric acid

• Cationic = basic dyes:• methylene blue• crystal violet• safranin• malachite green

Differential Stain

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Differential Stain

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1. Gram Stain

Crystal violet stain

Iodine mordant

95% ethanol decolorizeror ethanol/acetone

Safranin counterstain

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2. Acid Fast Stain (Ziehl-Neelson)

• Paul Ehrlich in 1882; • Detecting tuberculosis and leprosy – causing organisms.

Carbolfuchsin stain

3% HCl declorizerin 95% ethanol

Methylene Blue counterstain

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3. Special Stain: Negative Stain• Cell size• Cell shape

• Indian Ink or acidic dyesfor background

• Crystal Violet or Methylene Blue for counterstain

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4. Special Stain: Flagellar Stain• Silver;• Very difficult and time consuming;

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5. Special Stain: Endospore Stain (Schaeffer-Fulton)

Heat-fix

Malachite Green stain

Steam for 5 min to make the endo- spore wall permeable

Rinse in water for 30 sec Decolor

Safranin counterstain