MICROSCOPY AND STAINING

Leeuwenhoek’s Investigation 2

3 Metric Units

4 Light Properties Wavelength

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

Effect of Immersion Oil 10

Disappearing Diamond 11

Diffraction 12

Diffraction 13

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

Same Image – Different Techniques 20

21 Electron Microscopy Transmission (TEM or EM) Scanning (SEM) Scanning Tunneling (STM) Atomic Force (AFM) Scanning Acoustic (SAM) virtual/virtual.html *animation

Freeze - fracture 22

SEM Shadow Casting 23

SEM freeze-etch 24

25 Electron Microscopy Images

26

STM 27

AFM 28

29 Microscopy Techniques Wet Mounts Smears Drop

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

Differential Stain 31

Differential Stain 32

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

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 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 Safranin counterstain