1. MICROSCOPY AND STAINING

2. Leeuwenhoek’s Investigation 2

3. 3 Metric Units

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

5. 5 Light Properties Reflection Transmission

6. 6 Light Microscopy Types Compound Bright Field

7. 7 Light Properties Resolution

8. 8 Wavelength/Resolution Interaction

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

10. Effect of Immersion Oil 10

11. Disappearing Diamond 11

12. Diffraction 12

13. Diffraction 13

14. 14 Microscopy — Dark Field

15. 15 Microscopy — Phase Contrast Dual Beam

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

17. 17 Microscopy — Fluorescence

18. 18 Microscopy — Confocal Confocal

19. 19 Microscopy Imaging Digital

20. Same Image – Different Techniques 20

21. 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 22

23. SEM Shadow Casting 23

24. SEM freeze-etch 24

25. 25 Electron Microscopy Images

26. 26

27. STM 27

28. AFM 28

29. 29 Microscopy Techniques Wet Mounts Smears Drop

30. Simple Stain 30 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 31

32. Differential Stain 32

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

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

35. 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. 36 4. Special Stain: Flagellar Stain Silver; Very difficult and time consuming;

37. 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 Safranin counterstain