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MICROSCOPY AND STAINING. Leeuwenhoek’s Investigation. Metric Units. Light Properties. Wavelength. http://eyetricks.com/0101.htm. Light Properties. Reflection Transmission. Light Microscopy Types. Compound Bright Field. Light Properties. Resolution. Wavelength/Resolution Interaction. - PowerPoint PPT Presentation
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MICROSCOPY AND STAINING
Leeuwenhoek’s Investigation
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3
Metric Units
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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