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Bacterial Staining

A.  Supravital Staining - Bacterium is killed during staining


B.  Vital / Intravital Staining - Bacterium remains alive


C.  Simple Stains

  1. Methylene blue, basic fuchsin
  2. Provide color contrast, impart same color to all bacteria

D. Differential Stains

1.  Impart different colors to different bacteria/bacterial structures


2.  Gram stain

  1. Originally devised by Christian Gram
  2. Primary staining with a pararosaniline dye: crystal violet, methyl violet, gentian violet
  3. Application of a dilute solution of iodine
  4. Decolourisation with an organic solvent e.g: ethanol, acetone, aniline
  5. Counterstaining with safranine
  6. Gram positive: violet, more acidic protoplasm, relative impermeability of the bacterial cell wall &     cytoplasmic membrane to the dye iodine complex
  7. Gram negative: red
  8. Not all or none phenomenon: gram positivity lost due to prolonged treatment with decolorizer or damage to cell wall. 

E.  Acid Fast Stain

  1. Discovered by Ehrlich
  2. Tubercle bacilli resist decolorization with acids after staining with aniline dyes
  3. Modified by Ziehl and Neelsen
  4. Smear stained with carbol fuchsin with application of heat
  5. Decolourised with acid
  6. Counterstained with methylene blue
  7. Acid fast bacteria appear red
  8. Due to high content of fatty acids, alcohols
  9. Acid fast structures: mycobacteria (M. tuberculosis & atypical mycobacteria 20% H2SO4; M. leprae 5% H2SO4; Nocardia 1% H2SO4; Cryptosporidium, isospora, Cyclospora 5% H2SO4; bacterial spores 0.25%H2SO4.

F.  Negative Stains

  1. India ink, Nigrosin
  2. Provide uniform colored background against which the unstained bacteria stand out in contrast
  3. Demonstration of bacterial capsule
  4. Demonstration of slender bacteria (spirochetes)  

G. Impregnation Methods

  1. Very slender structures not visible by ordinary microscope are thickened to render them visible by impregnation of silver on their surface
  2. Levaditi’s and Fontana's stain for spirochetes
  3. Leifson's & Ryu’s stain for flagella

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