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A. Important Points

  1. The diameter of the smallest body that can be resolved and seen clearly with naked eye: 200 μm
  2. Medically important bacteria:  0.2 - 1.5 μm diameter, 3 - 5 μm length
  3. Living state/ after fixation and staining
  4. Ordinary white light as source of illumination
  5. Wet film/hanging drop: arrangement, motility, size
  6. Resolving power: (1/2 the wave length): 0.1μm - 0.2μm
  7. Disadvantage: no internal details can be made out
  8. Light waves passing through transparent objects such as cells, emerge in different phase
  9. Special optical system converts the difference in phases into difference in intensity
  10. Reveals details of internal structure of living cells
  11. Reflected light is used instead of transmitted light
  12. Essential part: dark-ground condenser
  13. For visualization of very slender organisms: e.g. spirochetes (diameter < 0.2μm)
  14. When ultraviolet/invisible light fails on a fluorescent substance, the wavelength of the invisible light increases so that it becomes luminous
  15. Cells stained with fluorescent dyes: become luminous and seen as bright object against dark background
  16. Some fluorescent dyes have selective action for particular cell constituent eg :
    1. Auramine & Rhodamine:
      mycolic acid, mycobacteria appear bright yellow/orange against greenish background
    2. Acridine orange :  binds nucleic acid (bright orange)
    3. Calcofluor white : binds to cell wall (chitin) of fungi (blue white) 

B. Types of Microcopes

  1. Light Microscope
  2. Phase - Contrast Microscope
  3. Dark- Field Microscope
  4. Fluorescence Microscope
Extra Edge :

Fluorochromes in immunofluorescence microscopy
FITC – Fluorescein isothiocyanate (most commonly used)
TRITC-tetramethyl rhodamine isothiocyanate


5.  Immunofluorescence

Direct, Indirect


6.  Electron Microscope

  1. Beam of electrons used instead of beam of light
  2. Beam focussed by circular magnets instead of lenses
  3. The object held in the path of the beam scatters the electrons
  4. Image focussed on fluorescent viewing screen
  5. Wavelength of electrons: .005nm ; visible light : 500nm
  6. Theoretically EM should be 100,000 times more powerful
  7. In practice maximum resolution that can be obtained:
    i. 0.3-0.5nm (hundred times better than light microscope)
  8. Examination under vacuum

7.  Interference Microscope

  1. Reveals cell organelles
  2. Enables quantitative measurements of the chemical constituents of cell eg: lipids, proteins, nucleic acid

8.  Polarisation Microscope

  1. Enables the study of intracellular structures using differences in birefringence

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