Negative Staining for Bacterial Capsule Demonstration: Introduction, Principle, Procedure and Result Interpretation

Negative Staining for Bacterial Capsule Demonstration: Introduction, Principle, Procedure and Result Interpretation

Introduction of Negative Staining for Bacterial Capsule Demonstration

Negative staining is so called because the background gets stained and the capssule of organism remains colorless. e.g.  India ink , nigrosin or eosin are the stains used. It is applicable in following bacteria-

  • Streptococcus pneumoniae,
  • Klebsiella pneumonie,
  • Haemophilus influenzae,
  • Neisseria meningitides, etc.

Principle of Negative Staining for Bacterial Capsule Demonstration

Capsules of bacteria are non-ionic and therefore stain will not adhere to their surfaces. The best method to visualize them is to stain the background using an acidic stain (e.g., Nigrosine, Congo red) and to stain the cell itself using a basic stain (e.g., crystal violet, safranin, methylene blue and basic fuchsin).

Requirements for Negative Staining for Bacterial Capsule Demonstration

  • Nigrosin stain/ India ink
  • Clean and grease free slides and cover slip
  • Test organism suspected specimens e.g.CSF from bacteria meningitis, growth of bacteria on solid media
  • Inoculating loop
  • Bunsen burner
  • Microscope
  • Cedar wood oil ( optional for bacteria)

Procedure of  Negative Staining

  1.  Transfer a aseptically a loopful of culture on a clean and dry slide.
  2.  Mix it with a loopful of stain either nigrosin or India ink.
  3. Make a smear by using a glass slide.
  4. Allow in to dry slowly at room temperature.
  5. Oberve under the microscope at 100X objective i.e. oil immersion lens.

Result Interpretation of  Negative Staining

In-case of nigrosin preparation

Background : Bluish black

Organisms: Colorless ( hollow bodies)

While in case of India Ink Preparation

background:  dark

Bacterial cell: purple

Capsule: Clear against the dark background ( as shown above picture)

Keynotes on Negative Staining

  1. India ink method contains two dyes (crystal violet and India ink). The capsule is seen as a clear halo around the organism against the black background. The background will be dark is due to colour of India ink. The bacterial cells will be stained purple since bacterial cells take crystal violet-basic dyes as they are negatively charged). The capsule (if present) will appear clear against the dark background because of capsule does not take any stain.
  2. Nigrosin stain uses ion negative staining method permits visualization of the usually transparent and unstainable capsules of various micro- microorganisms  like Cryptococcus neoformans (most commonly), Klebsiella pneumonie, Streptococcus pneumoniae, etc.
  3. This stain uses in tests for viability because living cells exclude the dye, but it enters dead cells.
  4. Assessment of viability and motility parameters of sperms during semen alaysis: In vitro viability is generally assessed both before and after thawing by eosin–nigrosin stain.
  5. Composition of Nigrosin:Following  are the ingredients of nigrosin stain.
  • Nigrosin :  10.0 gm
  • Formalin:  0.5 ml
  • Distilled water:  100.0 ml

Further Reading

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