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MICROSCOPY AND STAINING

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.

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MICROSCOPY AND STAINING

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  1. MICROSCOPY AND STAINING

  2. Leeuwenhoek’s Investigation

  3. Metric Units

  4. Light Properties • Wavelength http://eyetricks.com/0101.htm

  5. Light Properties • Reflection • Transmission

  6. Light Microscopy Types • Compound Bright Field

  7. Light Properties • Resolution

  8. Wavelength/Resolution Interaction

  9. Light Properties • Absorption • Refraction http://interactagram.com/physics/optics/refraction/

  10. Effect of Immersion Oil

  11. Disappearing Diamond

  12. Diffraction

  13. Diffraction

  14. Microscopy — Dark Field

  15. Microscopy — Phase Contrast • Dual Beam

  16. Microscopy — DIC (Nomarsky) • Differential Interference Contrast

  17. Microscopy — Fluorescence

  18. Microscopy — Confocal • Confocal

  19. Microscopy Imaging • Digital

  20. Same Image – Different Techniques

  21. 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

  22. Freeze - fracture

  23. SEM Shadow Casting

  24. SEM freeze-etch

  25. Electron Microscopy Images

  26. Electron Microscopy Images

  27. STM

  28. AFM

  29. Microscopy Techniques • Wet Mounts • Smears • Drop

  30. Simple Stain • Provide basic contrast for light compound microscope • Anionic = acidic dyes: • eosin • picric acid • Cationic = basic dyes: • methylene blue • crystal violet • safranin • malachite green

  31. Differential Stain

  32. Differential Stain

  33. 1. Gram Stain Crystal violet stain Iodine mordant 95% ethanol decolorizer or ethanol/acetone Safranincounterstain

  34. 2. Acid Fast Stain (Ziehl-Neelson) • Paul Ehrlich in 1882; • Detecting tuberculosis and • leprosy – causing organisms. Carbolfuchsin stain 3% HCldeclorizer in 95% ethanol Methylene Blue counterstain

  35. 3. Special Stain: Negative Stain • Cell size • Cell shape • Indian Ink or acidic dyes • for background • Crystal Violet or Methylene • Blue for counterstain

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

  37. 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 Safranincounterstain

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