Indirect (negative) staining

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Indirect (negative) staining

• Most biological materials show little contrast
  with their surroundings unless they are stained.
• In the case of light microscopy, contrast can be
  enhanced by using colored stains which
  selectively absorb certain wavelengths.

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Indirect (negative) staining

• To heighten the contrast between bacteria and the
  background, use is made of electron-dense
  "stains".
• These are usually compounds of heavy metals of
  high atomic number, that serve to scatter the
  electrons from regions covered with the stain.
• If bacterial particles are coated with stain
  (positive staining), fine detail may be obscured.

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Indirect (negative) staining

• Negative staining overcomes this problem by
  staining the background and leaving the bacteria
  relatively untouched.
• The negative stain is molded round the bacterial
  particle, outlining its structure, and is also
  able to penetrate between small surface
  projections and to delineate them.

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Indirect (negative) staining

• The acidic dye nigrosine will be used to
  visualize the capsule or sheath that surrounds
  some bacteria in a process called negative
  staining.
• Capsules are composed primarily of
  polysaccharides or glycoproteins and are
  gelatinous in texture.

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Indirect (negative) staining

• They are readily destroyed by heating and hence
  direct staining methods cannot be utilized.
• In general, the size and shape of microorganisms
  is often less distorted with indirect staining
  procedures, especially when sampled from a broth
  culture

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Indirect (negative) staining

• Therefore negative staining is useful whenever
  the morphology of individual bacteria is in
  question.
• Morphology can often be determined with
  confidence with only the high dry lens. Consider
  that this procedure does not necessarily kill the
  organism, so be careful.

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Indirect (negative) staining

• After preparing a clean, greaseless slide, a
  small drop of nigrosine is mixed with a small
  drop from a broth culture or with a quantity of
  dry material.
• The drop is spread across the slide using the
  edge of another slide as a spreader. This same
  procedure is used for blood smears.

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